Osteomark® is a urinary assay that provides a quantitative measure of the excretion of crosslinked N-telopeptides of type I collagen (NTx) as an indicator of human bone resorption. Elevated levels of urinary NTx indicate elevated human bone resorption. Measurement of NTx is intended for use in:

A. Predicting skeletal response (bone mineral density) to hormonal antiresorptive therapy in postmenopausal women

B. Therapeutic monitoring of:

1. anti-resorptive therapies in postmenopausal women
1. anti-resorptive therapies in individuals diagnosed with osteoporosis
1. anti-resorptive therapies in individuals diagnosed with Paget's disease of bone
1. estrogen-suppressing therapies

The measurement range of Osteomark is 20 to 3000 nM Bone Collagen Equivalents (BCE) of NTx.

Device Description

Osteomark is a competitive enzyme-linked immunosorbent assay (ELISA) which utilizes a horseradish peroxidase labeled monoclonal antibody directed against the cross-linked Ntelopeptides (NTx) present in urine specimens. An Osteomark® kit is comprised of the following reagents:

Antigen Coated 96-Well Plate Calibrators: 1 nM BCE 30 nM BCE 100 nM BCE 300 nM BCE 1000 nM BCE 3000 nM BCE Antibody Conjugate Concentrate Antibody Conjugate Diluent Level I and Level II Urine Controls 30X Wash Concentrate Buffered Substrate Chromogen Reagent Stopping Reagent

The solid phase utilizes microwells onto which NTx has been adsorbed. NTx in the specimen or Calibrator competes with the solid phase NTx for antibody binding sites. The resulting amount of Antibody Conjugate bound to the solid phase is indirectly proportional to the amount of NTx in the specimen or Calibrator. The quantity of NTx in the specimen is determined from a standard calibration curve using reagents supplied in the kit. Assay values are standardized to an equivalent amount of bone collagen, and are expressed in nanomole bone collagen equivalents per liter (nM BCE). BCE reflects the amount of immunoreactive NTx, as measured by Ostcomark, liberated from human bone collagen following digestion with bacterial collagenase, as measured by hydroxyproline by high performance liquid chromatography (HPLC).

AI/ML Overview

Acceptance Criteria and Device Performance for Osteomark®

This response synthesizes information from the provided text to describe the acceptance criteria and the studies that prove the Osteomark® device meets these criteria.

1. Table of Acceptance Criteria and Reported Device Performance

Given that Osteomark is a diagnostic assay, acceptance criteria typically revolve around its analytical and clinical performance in fulfilling its intended use. Here's a table based on the provided data:

Acceptance Criterion	Reported Device Performance
Analytical Performance
Lower Limit of Detection (LLOD)	20 nM BCE
Intra-assay Variability	Average 8% CV (range 5-19% CV)
Inter-assay Variability	Average 4% CV (range 3-5% CV)
Antigen Recovery	Average 105% (over 200-2500 nM BCE range)
Dilutional Linearity	Correlation coefficients of r=0.999 to 1.000 (over 44-2940 nM BCE range)
Clinical Performance (Predicting Skeletal Response to HRT)
Sensitivity for >30% decrease in Osteomark predicting positive BMD response	80% (95% C.I. 70%, 88%)
Specificity for >30% decrease in Osteomark predicting positive BMD response	59% (95% C.I. 36%, 79%)
Predictive Value Positive (PVP) for 30% change (at 80% prevalence)	88.6%
Predictive Value Negative (PVN) for 30% change (at 80% prevalence)	42.4%
Association between baseline Osteomark and risk of BMD loss without HRT	High baseline Osteomark (≥67 nM BCE/mM creatinine) indicated 17.3 times higher risk of spine BMD loss without HRT.
Clinical Performance (Monitoring Estrogen Suppressing Therapy)
Mean increase in Osteomark during estrogen suppression	68% increase from baseline (correlated with -3.7% decrease in lumbar spine BMD)
Percentage of subjects with ≥30% increase in Osteomark during estrogen suppression	63% (55/88)
Clinical Performance (Monitoring Anti-Resorptive Therapy in Paget's Disease)
Clinically significant change (≥30%) in Osteomark achieved (all therapies)	Yes, at each timepoint (Month 1, 3, 6)
Correlation with total alkaline phosphatase	High positive correlation at baseline and 6 months (r=0.72-0.88, p=0.0001-0.0003)
Earlier assessment of therapeutic response compared to total alkaline phosphatase	Osteomark showed 19% responders at Month 1 vs. 2% for total alkaline phosphatase
Clinical Performance (Monitoring Anti-Resorptive Therapy in Osteoporosis)
Percentage of alendronate group with Osteomark < 35 nM BCE/mM creatinine at 3 months	80% (71/89)
Mean percent decrease from baseline at 3 months (alendronate group)	-62% (± 20)
Percentage of alendronate group with > 40% decrease at 3 months	87% (76/87)

2. Sample Sizes and Data Provenance

The document provides details for several studies:

Reference Range Determination (Premenopausal Women):
- Sample Size: 258 women
- Data Provenance: Multi-center, cross-sectional study; likely prospective for urine collection after study definition.
Reference Range Determination (Men):
- Sample Size: 81 men
- Data Provenance: Study conducted at a large reference laboratory. Implied prospective since it's for reference range establishment.
Within-Subject Variability:
- Sample Size: 8 healthy subjects
- Data Provenance: Urine specimens collected every 2-3 days over 2 months. Prospective.
Intra-assay Variability:
- Sample Size: 8 urine specimens, each tested in replicates of 10.
- Data Provenance: Not specified, likely internal lab testing.
Inter-assay Variability:
- Sample Size: 3 urine specimens, each tested in duplicate over 20 separate assay runs.
- Data Provenance: Not specified, likely internal lab testing.
Antigen Recovery:
- Sample Size: 3 normal urine specimens.
- Data Provenance: Not specified, likely internal lab testing.
Dilutional Linearity:
- Sample Size: 4 urine specimens.
- Data Provenance: Not specified, likely internal lab testing.
Clinical Study: Predicting Skeletal Response & Monitoring HRT in Postmenopausal Women:
- Sample Size (completed study): 227 women (109 HRT group, 118 calcium group)
- Data Provenance: Multi-center, randomized, prospective clinical trial. (Campodarve et. al., 1995)
Clinical Study: Monitoring Estrogen Suppressing Therapy:
- Sample Size: Not explicitly stated for all analyses, but for percent change analysis: 88 subjects (55/88 had ≥30% change, 33/88 had <30% change).
- Data Provenance: Multi-center, non-randomized, prospective, longitudinal clinical trial. (Marshall et. al., 1996)
Clinical Study: Monitoring Anti-Resorptive Therapy in Paget's Disease:
- Sample Size: 72 subjects at baseline, varying slightly by timepoint and analysis (e.g., N=22, 17, 20 for Month 1 by therapy).
- Data Provenance: Not explicitly stated as multi-center, but implies a clinical research study. Prospective.
Clinical Study: Monitoring Anti-Resorptive Therapy in Osteoporosis (Alendronate Trial):
- Sample Size: Alendronate group: 91 at baseline, varying to 78 at Month 36. Calcium-only group: 188 at baseline, varying to 149 at Month 36.
- Data Provenance: Multi-center, randomized, prospective study. (Liberman et. al., 1995)

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the number of experts or their qualifications for establishing ground truth, except implicitly through the nature of the studies:

For BMD measurements (DEXA): Implies standard clinical practice for interpreting DEXA scans, likely by radiologists or trained technicians/clinicians.
For Paget's disease diagnosis: Based on "radiographic evidence and a serum total alkaline phosphatase level at least twice the upper limit of normal," implying diagnoses made by specialists (e.g., endocrinologists, rheumatologists) who interpret these results.
For osteoporosis diagnosis: Based on "lumbar spine bone mineral density ≥ 2.5 SD below the mean for mature premenopausal women," implying diagnosis by specialists.
For hormonal assays (estradiol): Standard laboratory values and their interpretation.

4. Adjudication Method for Test Set

The document does not describe any specific adjudication method (e.g., 2+1, 3+1) for establishing ground truth in any of the studies mentioned. The clinical studies appear to rely on standard clinical diagnostic criteria and objective measurements (DEXA, serum markers).

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study is described in the provided text. The studies focus on the performance of the Osteomark® assay itself, either in isolation or in conjunction with clinical outcomes, rather than evaluating human readers' performance with and without AI assistance (which would not be applicable for an in vitro diagnostic assay like Osteomark®).

6. Standalone (Algorithm Only Without Human-in-the-Loop) Performance

Yes, the performance characteristics and clinical studies describe the standalone performance of the Osteomark® assay (the "algorithm only"). It's an in vitro diagnostic test where the output (Osteomark® value) is generated by the assay itself, and then interpreted by a clinician. There is no human-in-the-loop directly interacting with the algorithm's output generation process in the sense of an AI-assisted diagnostic system where a human reader's interpretation is augmented or compared to the AI.

7. Type of Ground Truth Used

The ground truth used varies by the type of study:

Analytical Performance: Based on known concentrations (e.g., calibrated standards for LLOD, antigen recovery, dilutional linearity) and statistical measures of precision (CV).
Predicting Skeletal Response to HRT:
- Bone Mineral Density (BMD) changes: Measured by Dual Energy X-ray Absorptiometry (DEXA).
- Response to HRT: Defined as "maintenance or gain in BMD."
Monitoring Estrogen Suppressing Therapy:
- Serum Estradiol Levels: Direct measurement of the therapy's effect on estrogen.
- Lumbar Spine (L1-L4) BMD changes: Measured by DEXA.
Monitoring Anti-Resorptive Therapy in Paget's Disease:
- Normalization of Serum Total Alkaline Phosphatase: A widely accepted clinical marker for Paget's disease activity and therapeutic response.
- Radiographic evidence: For initial diagnosis.
Monitoring Anti-Resorptive Therapy in Osteoporosis:
- Lumbar Spine Bone Mineral Density (BMD): Specifically, ≥ 2.5 SD below the mean for mature premenopausal women for baseline diagnosis.

8. Sample Size for the Training Set

The document does not explicitly delineate a "training set" in the context of machine learning or AI algorithm development. Osteomark® is described as a competitive ELISA assay, indicating it is a biochemical test, not an AI algorithm. Therefore, the concept of a separate training set for algorithm development (beyond the analytical validation and clinical validation described) does not apply in the typical sense. The "training" of the assay involves the establishment of the standard calibration curve using the provided calibrators.

9. How the Ground Truth for the Training Set Was Established

As Osteomark® is a competitive ELISA assay and not an AI algorithm, the concept of a "training set" with ground truth in the AI sense is not applicable. The assay's "calibration" or "standardization" is achieved through:

Calibrators: The kit includes six calibrators with known concentrations (1 nM BCE to 3000 nM BCE). These calibrators are used to generate a standard curve, which is essential for quantifying NTx in patient samples.
Standardization to Bone Collagen: Assay values are standardized to an equivalent amount of bone collagen, and expressed in nM BCE, reflecting immunoreactive NTx liberated from human bone collagen following digestion with bacterial collagenase, as measured by hydroxyproline by HPLC. This process ensures the assay measures the intended analyte accurately and consistently.

Summary

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Summary of Safety and Effectiveness

Antigen Coated 96-Well Plate Calibrators: 1 nM BCE 30 nM BCE JUL 11 1996 100 nM BCE 300 nM BCE 1000 nM BCE 3000 nM BCE Antibody Conjugate Concentrate Antibody Conjugate Diluent Level I and Level II Urine Controls 30X Wash Concentrate Buffered Substrate Chromogen Reagent Stopping Reagent

10 SIDET Sin

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Intended Use of Osteomark®

A. Predicting skeletal response (bone mineral density) to hormonal antiresorptive therapy in postmenopausal women

B. Therapeutic monitoring of:

1. anti-resorptive therapies in postmenopausal women
1. anti-resorptive therapies in individuals diagnosed with osteoporosis
1. anti-resorptive therapies in individuals diagnosed with Paget's disease of bone
1. estrogen-suppressing therapies

The measurement range of Osteomark is 20 to 3000 nM Bone Collagen Equivalents (BCE) of NTx.

Expected Values

Urine Collection:

A multi-center, cross-sectional study was conducted to determine the reference range for normal premenopausal women (mean age 36 years, range 25-49). Male reference range was determined from a study conducted at a large reference laboratory (mean age 46 years, range 24-71). The mean, standard deviation, and the mean ± 2 standard deviation ranges for these two populations are presented in Table 1 below.

Table 1 - Expected Osteomark® Values for Premenopausal Women and Men

	Mean*	Std Dev	Range(mean ± 2 std dev)*	N
Women	35	15	5-65	258
Men	27	12	3-51	81

expressed as nanomoles BCE/millimole creatinine

rk® 510(k) Submission rex Insternational, Inc.

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In a separate study, the expected within-subject variability was determined from urine specimens from eight healthy subjects collected every 2-3 days over approximately 2 months. The average of the individual within-subject longitudinal variation was 19.3%. The average between-subject longitudinal variation was 38.3%.

Limitations of the Procedure

Lower Limit of Detection

The lower limit of detection of the Osteomark® assay is 20 nM BCE. This value represents a concentration which is greater than the value which can be distinguished from zero, and was calculated by subtracting 3 standard deviations optical density (A450-A630) from established variability of the InM BCE Calibrator. Assay precision below this value is insufficient for accurate results.

Interfering Substances

Common urine components and contaminants, as well as microbiological contaminants, that are known to interfere with many laboratory urine analyses were evaluated for an interfering effect with Osteomark®. The evaluations were performed by adding normal and excessive quantities of each potential inhibitor to normal urine specimens and analyzing for an effect on the final results. Results show that specimens obviously contaminated with whole blood or that have extensive hemolysis may show interference in the assay. These specimens should be avoided, and the specimen recollected.

Limitations of the Procedure

While Osteomarks is used as an indicator of bone resorption, use of this test has not been established to predict development of osteoporosis or future fracture risk. Use of this test has not been established in primary hyperparathyroidism or hyperthyroidism. When using Osteomark® to monitor therapy, results may be confounded in patients afflicted with clinical conditions known to affect bone resorption, e.g., metastases to bone. While an

omark® 510(k) Submission Onex International, Inc.

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Osteomarks value provides a measure of the level of bone resorption, a single Osteomark® value can not provide the rate of bone resorption as reported results do not contain a measure of time. Osteomark@ results should be interpreted in conjunction with clinical findings and other diagnostic results.

Performance Characteristics

Reproducibility

Assay reproducibility was evaluated for intra-assay variability of normal urine specimens across the range of the calibration curve.

Intra-assay variability, or within assay precision, was assessed using eight urine specimens tested in replicates of 10 by each of four operators. Results demonstrate an average intraassay variability estimate of 8% CV, with a range of 5-19% CV along the calibration curve.

Inter-assay variability, or assay to assay precision, was assessed using three urine specimens tested in duplicate by one operator over 20 separate assay runs. Results demonstrate an average interassay variability estimate of 4% CV. with a range of 3-5% CV along the calibration curve.

Antigen Recovery

Antigen recovery was evaluated by adding known amounts of NTx to each of three normal urine specimens. Recovery represented the observed assay value of the "spiked" specimens, calculated as a percent of the expected urine value (baseline urine value plus added antigen value). Results demonstrate an average antigen recovery of 105%, over an assay value range of 200-2500 nM BCE.

toomark@ 510(k) Submission tex International, Inc.

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Dilutional Linearity

Dilutional linearity was evaluated by performing serial dilutions of four urine specimens with high nM BCE values into a urine specimen with a low nM BCE value. Results demonstrated correlation coefficients of r=0.999 to == 1,000 across an assay range of 44-2940 nM BCE.

Clinical Studies

Use of Osteomark® in Theraneutic Monitoring of Hornons) Anti-Resorntive Therany and in Predicting Skeletal Response (Bone Mineral Density) in Postmenonausal Women

A multi-center, randomized, prospective clinical trial was conducted to determine the ability of the Osteomark® assay to monitor the effect of hormonal anti-resorptive therapy on bone resorption in early postmenopausal women, and to predict response to hormone replacement therapy (HRT). Subjects were randomized to estrogen (0.625 mg) and medroxyprogesterone (2.5 - 5 mg cyclic or continuous) plus a daily 500 mg calcium supplement (HRT group), or a daily 500 mg calcium supplement only (calcium group). A total of 227 women, 109 in the HRT group and 118 in the calcium group, completed the 12 month study (Campodarve et.al., 1995).

The following data support the clinical utility of Osteomark® to monitor hormonal anti-resorptive therapy in early postmenopausal women and to predict changes in bone mineral density (BMD) measured by dual energy x-ray absorptiometry (DEXA) in response to HRT, thereby identifying who will receive the greatest benefit from such therapy, and to identify those at risk for bone loss.

Osteomark® monitors the effect of therapy

Figure I provides the Osteomarkes values throughout the study. Figure 2 provides the percent change from baseline Osteomark® throughout the study.

· The baseline Osteomark® value in the two groups combined was 59 + 2.1 (mean ± sem) nM BCB/mM creatinine.
· In the HRT Group, the values decrease toward the premenopausal mean.

Oncomand 510(k) Submission Ostex International, Inc.

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Image /page/5/Figure/0 description: The image is a title for a figure. The title reads "Figure 1: Osteomark® Values Throughout the Study". The title is written in a bold, serif font.

Image /page/5/Figure/1 description: This image shows a graph titled "HET GROUP". The x-axis shows the time in terms of "Baseline", "1 Month", "4 Month", and "1 Year". The y-axis shows a scale from 0 to 200. The graph shows the distribution of data points at each time point.

Image /page/5/Figure/2 description: This image is a bar graph titled "CALCIUM GROUP". The x-axis represents time intervals: Newborn, 1 Month, 6 Months, and 1 Year. The y-axis represents a numerical scale from 0 to 200, with increments of 10. The graph displays the distribution of calcium levels within the group at each time interval.

Ossomark® 510(k) Submission Octor International, Inc.

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Image /page/7/Figure/0 description: The image is a bar graph comparing data at 1 month, 6 months, and 1 year. The y-axis ranges from -100 to 190. The data at 1 month is more spread out, ranging from -70 to 60. The data at 6 months and 1 year is more concentrated around 0.

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CALCIUM GROUP

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Baseline Osteomark® values and the percent change from baseline to 6 months are predictive of the change in BMD and/or response to HRT.

Figure 4 provides the baseline Osteomark® values and the percent change from baseline to six months stratified into quartiles, with the corresponding percent change in spine BMD after 1 year of HRT.

· Subjects in the highest quartile of Osteomark® values at baseline (67 188 nM BCE/mM creatinine), or with the greatest decrease from baseline to 6 months of therapy (-66 to -87%), showed the greatest gain in spine BMD in response to HRT.
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Image /page/9/Figure/0 description: Figure 4 shows the HRT Group values of baseline and percent change in Osteomark to six months stratified by quartile and corresponding percent change of L1-L4 BMD at 1 year. The figure provides information on the changes in bone mineral density (BMD) in the lumbar spine (L1-L4) over a one-year period. The data is stratified by quartile, which means the study population is divided into four groups based on their baseline Osteomark values.

Image /page/9/Figure/1 description: The image contains two bar charts that show the percent change in L1-L4 to one year. The top chart shows quartiles by osteomark at baseline, with Q1 (18-38) having a percent change of around 1.5, Q2 (38-51) having a percent change of around 2.4, Q3 (51-67) having a percent change of around 2.4, and Q4 (87-188) having a percent change of around 3.5. The bottom chart shows quartiles by percent change in osteomark to six months, with Q1 (192 to -22%) having a percent change of around 1.3, Q2 (-22 to -51%) having a percent change of around 0.8, Q3 (-51 to -66%) having a percent change of around 3.2, and Q4 (-66 to -87%) having a percent change of around 4.4.

Ortsomark® 510(k) Submission Outex International, Inc.

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Contingency table analysis showed that a >30% decrease at 6 months was significantly associated (p<0.005) with a positive BMD response to HRT (maintenance or gain in BMD) at 1 year. The binomial 95% confidence intervals for the sensitivity and specificity of using a 30% change in Osteomark® for predicting a response to HRT are: Sensitivity = 80% (95% C.I. 70%, 88%)

Specificity = 59% (95% C.I. 36%, 79%)

The results of using Baye's Rule to define the predictive value positive (PVP) and predictive value negative (PVN) of a 30% change in Osteomark for predicting response are tabulated below for a range of prevalence values. A prevalence of 80% was seen in this study. With higher prevalence rates, low specificity and PVN percent values are indicative of a low number of subjects in the negative response category.

Prevalence	PVP	PVN
60%	66.1%	66.3%
70%	82.0%	55.8%
80%	88.6%	42.4%
90%	94.6%	24.7%
99%	99.5%	2.9%

Figure 5 provides the linear regression analysis (y = -0.03x + 1.3), r = -0.34, p = 0.0003) of the percent change from baseline to 6 months Osteomark® and percent change from baseline to I year BMD in the HRT Group (R2 = 0.12)

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Image /page/11/Figure/0 description: Figure 5 shows the HRT Group's linear regression of percent change from baseline to 6 months Osteomark and percent change from baseline to 1 Year L1-L4 BMD. The figure is a title for a graph or chart. The title provides information about the data being presented in the graph or chart.

Image /page/11/Figure/1 description: This image is a scatter plot showing the relationship between L1-L4 BMD percent change to one year and Osteomark percent change to six months. The x-axis represents Osteomark percent change to six months, ranging from -100 to 200, while the y-axis represents L1-L4 BMD percent change to one year, ranging from -14 to 16. The plot includes a regression line, and the equation for the line is y = -0.03x + 1.3, with a correlation coefficient of r = -0.34 and a p-value of p = 0.0003.

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Figure 6 provides the average on-study Osteomark® value for the calcium group stratified into quartlies, with the corresponding percent change in spine BMD after 1 year.

· Subjects maintaining the highest Osteomark® values (>64 nM BCE/mM creatinine) had the greatest decrease in spine BMD.
Figure 6: Calcium Group - Average On-Study Osteomark® Values Stratified by Quartile and Corresponding Percent Change L1-L4 BMD at 1 Year

Image /page/12/Figure/3 description: The figure is a bar graph that shows the percent change in L1-L4 from baseline to one year. The x-axis shows the quartiles, Q1 (21-38), Q2 (38-49), Q3 (49-64), and Q4 (64-139). The y-axis shows the percent change L1-L4 from baseline to one year (+/- sem). The p-value for Q1 and Q2 is "ns", the p-value for Q3 is less than 0.02, the p-value for Q4 is less than 0.001, and the p-value between Q1 and Q4 is less than 0.005.

Quartiles of Average On-Study Osteomark (nM BCE/mM creatinine)

Ostoomark® 510(k) Submission Oran International, Inc.

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Table 1 compares the two treatment groups using Relative Risk analysis.

· A high baseline Osteomark® value (≥67 nM BCE/mM creatinine) indicated a 17.3 times higher risk of loss of spine BMD if not treated with HRT.
Table 1: Relative Risk of Loss of BMD Comparing Calcium and HRT Groups

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18-38	1.4	0.8 - 2.5
38-21	2.5	1.0 - 6.1
51-67	3.8	1.6 - 9.1
67-188	17.3	2.5 - 118.6

Use of Osteomark® to Monitor Estrogen Suppressing Therany

A multi-center, non-randomized, prospective, longitudinal clinical trial was conducted to determine the ability of the Osteomark® assay to monitor the effect of estrogen suppressing therapy on bone resorption in premenopausal women (Marshall et.al., 1996). Subjects were given GnRH-agonist therapy 3-6 months for treatment of varied gynecologic disorders.

The following data support the clinical utility of Osteomark® to monitor the effect of estrogen suppressing therapy.

Figure 7 represents the mean (± sem) Osteomark® values obtained throughout the study along with corresponding mean (± sem) estradiol values for each timepoint.

· The mean Osteomark® value at baseline was 44 ± 3 nM BCE/mM creatinine.
· The mean Osteomark® value while estrogen suppressed was 61 nM BCE/mM creatinine, a 68% increase from baseline.
· The mean serum estradiol level was 21 pg/mL during this time period.
· The Osteomark and estradiol values while estrogen suppressed were concordant with postmenopausal ranges of each analyte.

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Image /page/14/Figure/0 description: The image is a title for a figure. The title reads "Figure 7: Osteomark® Values and Serum Estradiol Levels (mean ± sem) During and After Estrogen Suppression Therapy". The title indicates that the figure will likely show data related to Osteomark values and serum estradiol levels. The data will be presented as mean ± sem, and will cover the period during and after estrogen suppression therapy.

Image /page/14/Figure/1 description: The image is a graph that shows the mean Osteomark and mean serum estradiol levels over time. The x-axis represents the time points: Baseline, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 month post, and 3 months post. The y-axis on the left represents the mean Osteomark levels, while the y-axis on the right represents the mean serum estradiol levels. The Osteomark levels are represented by a bar graph, and the estradiol levels are represented by a line graph. The graph also indicates the period of estrogen suppression therapy.

Ostcomark® 510(k) Submission Ostex International, Inc.

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Figure 8 provides percent change in Osteomark® from baseline throughout the study for each subject.

· Sixty three percent (55/88) of the subjects had a mean percent change from baseline of≥30% (p=0.025).
· Subjects exhibiting < 30% change (33/88, or 37%) had a mean baseline Osteomark® value that was higher (60 nM BCE/mM creatinine) than those with a > 30% change (35 nM BCE/mM creatinine), accounting for the lesser percent change in Osteomark® in these individuals.
· The average on-therapy Osteomark value for the < 30% change group was lower (51 nM BCE/mM creatinine) than the > 30% group (66 nM BCE/mM creatinine). The 30% change group also tended to lose less bone at the spine than those who had a ≥ 30% change.

Oscomark® 510(k) Submission
Ostex International, Inc.

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Image /page/16/Figure/0 description: This image shows a graph of percent change from baseline during and after estrogen suppressing therapy. The x-axis represents different months, including month 1, mean of months 2 to 6, post 1 month, and post 3 months. The y-axis represents the percent change from baseline, ranging from -80 to 400. The graph shows the change in estrogen levels over time.

Figure 8: Osteomark® Percent Change from Baseline During and After Estrogen
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Further analysis showed:

· The mean 68% increase from baseline Osteomark® correlated to a mean percent decrease at six months of -3.7% in lumbar spine (L1-L4) BMD (r = - 0.46, p<0.01). Three months after cessation of estrogen therapy, the mean Osteomark® value returned to baseline (44 nM BCE/mM creatinine) as serum estradiol levels returned to normal premenopausal levels.
· Lumbar spine BMD remained below baseline (-2.4%) at the 3 month post estrogen suppression therapy timepoint.

Use of Osteomark@ in Therapeutic Monitoring of Anti-Resorptive Therapy in Paget's Disease of Bone

Paget's disease of bone is characterized by abnormally elevated rates of bone resorption coupled with elevated bone formation. A study was conducted to determine the ability of Osteomark® to monitor the effect of bisphosphonate therapy on bone resorption in Paget's disease patients. Subjects (65% male, 81% caucasian) were diagnosed with Paget's disease based upon radiographic evidence and a serum total alkaline phosphatase level at least twice the upper limit of normal, and were treated with one of three bisphosphonates; alendronate 40 mg/day, orally (n=28), etidronate 400 mg/day, orally (n=24), and pamidronate 60 mg intravenous x3 doses (n=20). Serum and urine specimens were collected at baseline and 1, 3, and 6 months after initiation of therapy. The following data support the clinical utility of Osteomark® to monitor the effect of bisphosphonate therapy on the rate of bone resorption in patients diagnosed with Paget's disease of bone.

omark® 510(k) Submission Ostex International. Inc.

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Table 2 provides Osteomark® values at each timepoint for the three treatment groups combined.

. Table 2: Osteomark @ Values (nM BCE/mM creatinine) at Baseline and On Therapy

Visit	Mean	sem
Baseline	1195	155
Month 1	599	87
Month 3	443	77
Month 6	307	63

(All Therapies Combined)

11 11 11

*N= 72, 59, 71, and 69 respectively

Table 3 provides the mean percent change from baseline Osteomark® for each therapy.

· At each timepoint and with all therapies, a clinically significant change in Osteomark® (≥ 30%) was achieved.
Table 3: Percent Change from Baseline Osteomark® by Therapy

Visit	Alendronate	Etidronate	Pamidronate
Month 1*	-48%	-39%	-71%
Month 3**	-77%	-58%	-67%
Month 6***	-87%	-72%	-71%

"N = 22, 17, and 20 respectively

**N = 28, 23, and 20 respectively

·**N = 27, 22, and 20 respectively

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Table 4 provides the Osteomark® values for responders and nonresponders at 6 months using total alkaline phosphatase normalization as the criteria for response.

The level of total alkaline phosphatase is often associated with the severity of . Paget's disease of bone. Normalization of total alkaline phosphatase levels has been used to determine response to therapy. Using linear regression of the logtransformed baseline Osteomark® and total alkaline phosphatase levels, a significant positive relationship was found in all three treatment groups (1-0.88, 0.74, and 0.87 in the alendronate, etidronate and pamidronate groups respectively; p=0.0001 for all groups). Thus patients with high levels of total alkaline phosphatase also had high levels of Osteomark® at baseline. Again at six months, the correlations of Osteomark and total alkaline phosphatase levels were high, 1 = 0.88, 0.87, and 0.72 and p=0.0001, 0.0001 and 0.0003 in the alendronate, etidronate and pamidronate groups respectively.

Table 4. Six Month Osteomark® Values (mean + SD) for Responders and Nonresponders Based on Normalization of Serum Total Alkaline Phosphatase

	Alendronate	Etidronate	Pamidronate
Responder	$31 \pm 36$	$32 \pm 27$	$66 \pm 37$
Nonresponder	$795 \pm 867$	$373 \pm 355$	$487 \pm 759$

*N = 18, 2, and 9 respectively

· * N = 9, 20, and 11 respectively

{20}------------------------------------------------

Table S provides the percentage of responders (all therapies combined), as defined by the normalization of the marker value (Osteomark® 5-65 nM creatinine, total alkaline phosphatase 39-117 IUIL).

A decrease of serum total alkaline phosphatase into its normal range has been . used as an indicator of therapeutic response. An analogous examination of Osteomark® values over time indicates a similar utility as a measure of response.
Osteomark provides an earlier assessment than total alkaline phosphatase of . therapeutic response, defined by normalization of values, to bisphosphonate therapy.

Table S. Percent Responders as Defined by Normalization of Marker (number of patients with normalized marker value/total number of patjents)

Visit	Osteomark Responders	Total Alkaline PhosphataseResponders
Month 1	19%	2%
Month 3	34%	28%
Month 6	42%	42%

*N = 59, 71, and 69 respectively

{21}------------------------------------------------

Use of Ostenmark® to Theraneutic Monitoring of Anti-Resorntive Therany in Osteonorosis

A multi-center, randomized, prospective study was conducted to establish the safety and efficacy of a new amino-bisphosphonate (alendronate) in the treatment of osteoporosis (Liberman et.al. 1995). Study subjects were postmenopausal women, age 45 to 80 years diagnosed with osteoporosis (lumbar spine bone mineral density ≥ 2.5 SD below the mean for mature premenopausal women), randomized to receive either alendronate 10 mg plus calcium supplements (500 mg daily) (alendronate group), or placebo and calcium supplements only (calcium group). Fasting second morning void urine specimens were collected at baseline and periodically throughout the three year study. The following data support the clinical utility of Osteomarks to monitor the rate of bone resorption in osteoporotic women treated with antiresorptive therapy (alendronate 10 mg).

Table 6 provides the Osteomark® values for the two groups.

· Three months after initiating treatment 80% (71/89) of the subjects in the bisphosphonate group had an Osteomark® value < 35 nM BCE/mM creatinine. The mean (+ SD) percent change from baseline at 3 months was -62% (+ 20), 87% (76/87) had a > 40% decrease.
· After I year of therapy, the mean percent change from baseline Osteomark® remained similar to the values at 3 months (-65% ± 18); with 90% (77/86) ≤ 35 nM BCE/mM creatinine and 92% (77/84) of the subjects having a > 40% decrease,
· In the calcium only group, the Osteomark® values remained relatively constant, with a mean -13% ± 37 change (mean ± SD) from baseline at 1 year.

ark $10(k) Submission Ostex International, Inc.

{22}------------------------------------------------

Table 6. Osteomark® Values (nM BCE/mM creatinine) in Osteoporotic Patients

Treated with Alendronate 10 mg plus Calcium Supplement or Calcium Supplements

Only (mean ± SD)

Treatment Group	Baseline	Month 1	Month 3	Month 6	Month 12	Month 24	Month 36
Alendronate 10mg andCalcium*	$70 \pm 33$	$31 \pm 21$	$25 \pm 16$	$22 \pm 13$	$22 \pm 13$	$20 \pm 9$	$18 \pm 9$
Calcium only**	$69 \pm 33$	$57 \pm 28$	$58 \pm 26$	$54 \pm 26$	$54 \pm 25$	$52 \pm 21$	$56 \pm 20$

"N = 91, 88, 89, 88, 86, 80, and 78 respectively

*** = 188, 182, 183, 180,174, 157, and 149 respectively

Ostoomark® 510(k) Submission Ostex International, Inc.

{23}------------------------------------------------

Figure 9 provides the percent change from baseline Osteomark® through 1 year of the study

Figure 9. Percent Change From Baseline Osteomark® Through 1 Year

romark® 510(k) Submission Ostex International, Inc.

{24}------------------------------------------------

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{25}------------------------------------------------

Image /page/25/Figure/0 description: This figure shows the percent change from baseline for the Alendronate group at 1 month, 6 months, and 1 year. The y-axis shows the percent change from baseline, ranging from -100 to 160. At 1 month, the percent change from baseline ranges from -90 to 20, at 6 months, the percent change from baseline ranges from -95 to 10, and at 1 year, the percent change from baseline ranges from -90 to 15.

ALENDRONATE GROUP

Regulation Number and Section

§ 862.1400 Hydroxyproline test system.

(a)
Identification. A hydroxyproline test system is a device intended to measure the amino acid hydroxyproline in urine. Hydroxyproline measurements are used in the diagnosis and treatment of various collagen (connective tissue) diseases, bone disease such as Paget's disease, and endocrine disorders such as hyperparathyroidism and hyperthyroidism.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 862.9.