Overcoming resistance to bisphosphonates through the administration of alfacalcidol: results of a 1-year, open follow-up study.

Abstract

This study intended to determine whether the replacement of vitamin D3 with alfacalcidol results in any bone mineral density (BMD) increase in 76 patients unresponsive to the combination of alendronate and conventional vitamin D3 treatment. In these patients the conventional vitamin D3 had been replaced with alfacalcidol (0.5 μg/day), and then the patients were followed up for a year. After treatment for 1 year, Wilcoxon test revealed a small but statistically significant (P < 0.001) increase in the BMD values of the forearm and lumbar vertebrae, in the serum calcium and urinary calcium/creatinine ratio in first-voided morning urine. However, the serum alkaline phosphatase activity, phosphorus, parathormone, osteocalcin levels and the urinary d-pyr/creatinine ratio decreased significantly (P < 0.001). As suggested by our results, combination therapy with alendronate and alfacalcidol increases bone density and improves the biochemical markers of bone turnover, without any substantial increase in the incidence of adverse effects.

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Overcoming resistance to bisphosphonates through the administration of alfacalcidol:
results of a one-year, open follow-up study


János Gaál, Tamás Bender, József Varga, Irén Horváth,
Judit Kiss, Péter Somogyi, and Péter Surányi



János GAÁL MD, Ph.D. (correspondent), Irén HORVÁTH MD, Judit KISS MD, and Péter
SURÁNYI MD., Ph.D.
‘Kenézy Gyula’ Hospital, Department of Rheumatology and Physical Therapy, H-4043 Debrecen,
BartókBéla u. 2-26., e-mail: gaalja@freemail.hu, Tel: +36 30 219 2021, Fax: +36 52 511 833
Tamás BENDER MD., Ph.D.
Polyclinic of the Hospitaller Brothers of St. John of God in Buda, Budapest, Hungary
József VARGA, Ph.D.
University of Debrecen, Department of Nuclear Medicine, Debrecen, Hungary
Péter SOMOGYI MD.
Semmelweis University, Faculty of Medicine, Department of Orthopaedics, Budapest, Hungary

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Introduction
Osteoporosis is a disorder that leads to enhanced bone fragility through a decrease in the mass
as well as deterioration of the microarchitecture of bones. Owing to its high prevalence and
negative impact on quality of life, osteoporosis is the third most important public health
disorder (after malignancies and cardiovascular disease), afflicting 200 million people
worldwide [1]. Oral bisphosphonates are routinely prescribed for treatment for patients with
osteoporosis. Adequate calcium and vitamin D sufficiency of the body is essential for
antiresorptive treatment to be effective. According to HORVÁTH et al., serum 25-
hydroxyvitamin D3 level is lower than normal in 40% of elderly nursing home residents,
whereas data from BHATTOA et al. reflect the same in 56.7% of postmenopausal women [2,
3]. Some studies demonstrated statistically significant reduction of fractures during five years
of high dose (100 000 E every four months) treatment with vitamin D3 [4], but others not [5-
7]. According to a recent study, in 8 to 25 per cent of osteoporotic patients, bone loss
continues despite adequate treatment with bisphosphonates, administered in combination
calcium and vitamin D3 supplementation [8]. A proportion of these patients may have
functional hypovitaminosis D potentially resulting from insufficient intake (despite
supplementation), impaired activation, genuine resistance to vitamin D3 or secondary
hyperparathyroidism [9]. In the remainder, the cause underlying the lack of bisphosphonate
effects is unknown.
In osteoporosis, genuine resistance to bisphosphonates is probably non-existent or may be
considered extremely uncommon, at the least. In Paget’s disease, by contrast, where therapy is
aimed at achieving complete biochemical remission, resistance to bisphosphonates has been
demonstrated by several researchers. The lack of a complete biochemical remission is
regarded by some as the criterion for bisphosphonate resistance [10], whereas according to
others [11], the latter is established by a lower than 50-per-cent decrease in serum alkaline
phosphatase (AP) level. Notwithstanding the criteria, the prevalence of resistance varies with
different bisphosphonates in the range between 83% (etidronate [10]) and 11% (zolendronate
[12]).
According to additional, relevant evidence, the efficacy of activated analogues of vitamin D
(alfacalcidol, calcitriol) in reducing bone fractures and increasing bone density is superior to
that of native vitamin D. This has been demonstrated by several studies in various disease
forms, i.e. in postmenopausal osteoporosis, as well as in osteoporosis related to inflammatory
joint disease, or glucocorticoid therapy [13-16].

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This study was intended to ascertain:
1. whether replacing vitamin D3 with alfacalcidol is followed by an increase in BMD (i.e.
bisphosphonate resistance can be overcome) in patients not responding to treatment with a
bisphosphonate administered in combination with supplemental calcium and conventional
vitamin D3;
2. the incidence of hypovitaminosis D3 in the study population;
3. the changes occurring in biochemical markers of bone turnover during one year of
treatment with alfacalcidol,
4. the frequency of adverse events associated with the use of alfacalcidol.

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Patients and methods
Patients
Seventy-six patients were enrolled into the study conducted between January 2006 and March
2007. The duration of follow-up was 12 months on average. Inclusion criteria were:
diagnosed postmenopausal or senile osteoporosis (in females) or idiopathic osteoporosis in
males, and greater than 3% decrease in BMD – as demonstrated by axial DEXA – despite
adequate treatment (vitamin D3 400-1000 U/day, calcium 1000 mg/day and alendronate
70 mg/week) for a year or longer. Exclusion criteria comprised established secondary
osteoporosis, other forms of calcipenic osteopathy, hypercalcaemia, and history of renal
calculosis. The male-to-female ratio was 4:72; mean age of the study population was 70.5
(±8.2) years. Ten patients had a history of at least one prevalent vertebral or low-trauma
peripheral fracture. The average serum 25-hydroxyvitamin D3 level was 72,2± 36,2 nmol/l at
the starting and 74,1 ± 34,0 nmol/l at the end of the study (the difference was not significant
statistically). Below normal serum 25-hydroxyvitamin D3 level (<75 nmol/l) was found in 38
patients, along with elevated (>72 pg/ml) serum PTH level in 23 of these subjects. The mean
glomerular filtration rate (GFR) was 85,3 ml/min/1,73m2 at baseline and 4 patients had GFR
lower than 60 ml/min/1,73 m2 at starting of the study, 2 of them had elevated serum PTH
level. In the included patients, the average administered dose of D3 vitamin was 530 U/day
along with 1000 mg of calcium supplementation before the starting of the study.
Study design
During the follow-up period of one-year on average, control visits were scheduled at baseline
and at 3-month intervals thereafter. At baseline, a detailed history (concomitant diseases,
fractures, osteoporosis and risk factors) was recorded along with appraisal of clinical status by
obtaining a lateral x-ray of the lumbosacral spine and performing a laboratory screen (ESR,
CRP, CBC, serum calcium, phosphorus, BUN, creatinine, GOT, GPT, AP, albumin,
osteocalcin, 25-hydroxyvitamin-D3, parathormon [PTH] levels, urinary calcium/creatinine
[UCa/Cr] and deoxypyridinoline crosslinks/creatinine [D-Pyr/creatinine] ratios). Bone density
was measured in both peripheral bones (DTX200) and in the axial skeleton (DEXA, Lunar
DPX Pro) and BMD values obtained from the radius distal segment, as well as the means of
the values measured in the 1st through 4th lumbar vertebrae were taken into account. Serum
calcium level and urinary calcium/creatinine ratio were determined at control visits repeated
at 3-month intervals. After one year, information was gathered on fractures and renal

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calculosis that had occurred during the follow-up period, as well as osteodensitometry,
lumbosacral x-ray, and laboratory screen were repeated.
Treatment protocol
The pre-existing regimen of alendronate 70 mg once per week was left unchanged, but
conventional vitamin D3 and calcium treatment was replaced with 0.5 µg/day alfacalcidol (1α-
hydroxvitamin D3). The calcium supplementation was stopped also.
Endpoints
The primary endpoint of the study was defined as the change measurable (by DEXA) in bone
mineral density (BMD) of the forearm and of axial bones one year after the introduction of
alfacalcidol treatment. Secondary endpoints included changes in clinical chemistry parameters
(serum calcium, phosphorus, and alkaline phosphatase levels), biochemical markers of bone
turnover (serum osteocalcin, PTH, urinary D-Pyr/creatinine), UCa/Cr ratio determined in
first-voided morning urine, treatment-emergent adverse reactions, and osteoporotic fractures
that occur during follow-up.
Statistical analysis
The differences between the values of study parameters (BMD; serum Ca, P, AP, osteocalcin,
PTH levels; urinary D-Pyr/creatinine and UCa/Cr ratios) determined at two time points were
analysed using the SPSS 15.0 software package. First, the normality of differences was
checked with the Kolmogorov-Smirnov test (normal distribution would allow performing a
parametric [t or d] test). The differences between the paired values of all nine variables were
significantly different from the normal distribution (p<0.01 for UCa/Cr and p<0.001 for the
rest). As the data were unsuitable for analysis with the t-test, non-parametric Wilcoxon
signed-rank test was performed to check the uniformity of repeated measurements. (This
method is a non-parametric alternative to performing a paired t-test.)

Results
a) Changes in BMD during follow-up
The median of changes as well as the minimal and maximal changes are summarised in
Table 1.

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Unit
Median
Minimum
Maximum
Se Calcium mmol/l
0.06
-0.09 1.13
Se Phosphate mmol/l
-0.05
-0.49 0.3
Alkaline phosphatase U/l
-13
-46 287
Parathormone pg/ml
-10.7
-150 22.5
Urinary Ca/creatinine
0.1
-0.32 0.49
Osteocalcin ng/ml
-0.4
-5.8 4.2
BMD of forearm g/cm²
0.007
-0.011 0.039
BMD of lumbar 1-4
vertebrae
g/cm²
0.012
-0.998 0.116
Relative change of forearm
BMD
%
2.18
-3.5 16.3
Relative change of lumbar
BMD
%
1.38
-11.3 15.8
Deoxypyridinoline
crosslinks/creatinine
nmol/mmol
-0.2
-15.69 0.12

Table 1: Median, minimal and maximal changes in various parameters as a result of treatment

After treatment for 396,5 ± 21,3 days with alendronate – with no change in the original
dosage regimen of 70 mg/week, but – in combination with 0.5 µg/day alfacalcidol instead of
the previously prescribed conventional vitamin D3 (administered with calcium), we observed
the following changes (median values): forearm BMD increased by 0.007 g/cm², and lumbar
(L1-4) BMD increased by 0.012 g/cm². The changes in T-scores showed rather high scatter.
The relative changes in BMD values were 2.18% and 1.38% for the forearm and lumbar
vertebrae, respectively. BMD changes that have occurred during one-year follow-up are
shown in Figure 1. Box and whiskers plots are summarizing the median, quartiles, and
extreme values. The box represents the interquartile range which contains the 50% of values.

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The whiskers are lines that extend from the box to the highest and lowest values, excluding
outliers. A line across the box indicates the median.
Compared to baseline values, densitometry results (both BMD values and T-scores) obtained
after one-year treatment revealed statistically significant improvement in both regions
(Wilcoxon’s signed rank test, p<0.001).
b) Changes in clinical chemistry parameters
At baseline, mean serum calcium level was 2.34 (±0.106) mmol/l, phosphorus level was 1.18
(±0.169) mmol/l, and UCa/Cr ratio measured in first-voided morning urine was 0.25 (±0.161).
After one year of treatment, serum calcium level increased by 0.06 mmol/l (median), while
serum phosphorus level decreased by 0.05 mmol/l. Urinary Ca to creatinine ratio measured in
first-voided morning urine increased by 0.1 (median). All these changes were significant
(Wilcoxon’s signed rank test, p<0.001) (see Figure 2). The decline of serum alkaline
phosphatase level over the follow-up period by a median of 13 U/l was also significant, but
clinically not relevant (see Figure 3).
c) Changes in biochemical markers of bone turnover
Changes in serum parathormone (PTH) level were as expected. At baseline, elevated PTH
was ascertained in 32 patients; this number decreased to 16 during the one-year follow-up.
The median change in serum PTH level was significantly lower one year later (median
change: -10.7 pg/ml) (Figure 3). Serum osteocalcin level decreased by 0.4 ng/ml (median)
after treatment; this was accompanied by a decrease in the D-Pyr/creatinine ratio by 0.2
nmol/mmol (p<0,001) (Figure 3). Although the changes showed high scatter, they proved to
be highly significant (Wilcoxon’s signed rank test, p<0.001). The ranges of relative changes
expressed as percentages of the base values for all parameters (Figure 4).
d) Adverse events
Mild hypercalcaemia (serum calcium level <3 mmol/l) was observed in 3 patients; no other
clinically relevant adverse reactions were seen. There was no significant increase in urinary
calcium based on alendronate therapy.
e) New fractures
As stated above, ten patients had a history of at least one prevalent vertebral or low-trauma
peripheral fracture at starting of the study. Four new vertebral and two peripheral (wrist)

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fractures were recorded during follow up. Two of the vertebral fractures were sustained by
patients with a prevalent vertebral fracture.
Discussion
Some data from the literature suggest that activated vitamin D derivatives (alfacalcidol,
calcitriol), also called D-hormone analogs, are superior to conventional vitamin D, as regards
the mitigation of fracture risk. In their meta-analysis of 33 clinical studies, RICHY et al. found
a 13.4-per-cent (delta RD, 95% CI 7.7-19.8) reduction of fracture risk during the use of
activated vitamin D3 derivatives, compared to a mere 6% (delta RDi, 95% CI 1-12)
accomplished by treatment with conventional vitamin D3 [ 13]. The rate difference (RD) was
statistically significant (ANOVA-1; P< 0.001) RINGE et al. studied the changes of BMD and
bone fractures in patients with glucocorticoid-induced osteoporosis, over 3 years of treatment
with either alfacalcidol or vitamin D3 – both administered with calcium supplementation of
500 mg daily. Changes in the BMD of the lumbar spine were 2.4% vs. -0.8% (p<0.0001), and
of the femoral neck 1.2% vs. 0.8% (p<0.006), respectively. The proportion of patients who
have sustained at least one new vertebral fracture was 9.7% in the alfacalcidol and 24.8% in
the control group (risk reduction: 0.61, p=0.005), whereas the proportions of patients with at
least one new non-vertebral fracture were 15% vs. 25% (risk reduction: 0.41, p=0.08). Taking
the occurrence of any type of incident fractures into account, the above proportions were
19.4% vs. 40.6%, respectively (risk reduction: 0.52, p<0.001) [14].
During the AAC study published in 2007, 30-30 patients were randomised into either of the
following 3 treatment arms: a) alfacalcidol 1 µg + calcium 500 mg/day; b) alendronate
70 mg/week + calcium 1000 mg/day + vitamin D3 1000 IU/day; c) alendronate 70 mg/week +
calcium 500 mg/day + alfacalcidol 1 µg/day. During the 2 years of follow up, bone density of
the lumbar spine and of the total hip increased by 3%/1.5%, 5.4%/2.4%, and 9.6%/3.8%,
respectively. The magnitude of changes observed in patients treated with alendronate +
alfacalcidol + calcium was significantly greater than of those seen in the other two groups
(p<0.0001 for lumbar BMD and p<0.0002 for femur BMD). The number of new osteoporotic
(vertebral + non-vertebral) fractures occurring over 2 years of treatment in the three treatment
groups was 9, 10, and 2, respectively – this finding is also in support of the favourable effect
of alfacalcidol and especially of the combination of alendronate + alfacalcidol on fracture
prevention [16].
As suggested by several studies, administering antiresorptive agents (e.g. bisphosphonates)
with activated vitamin D3 derivatives may increase the success of therapy. The beneficial

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effects of alfacalcidol on intestinal calcium absorption, osteoid mineralization, muscle
function and motor coordination, serum PTH level, and the risk of falls may substantially
enhance the efficacy of treatment with bisphosphonates [17-18].
The anabolic effect of alfacalcidol on bone is supported also by in vivo and in vitro data.
Under the effect of alfacalcidol, osteoblasts have been shown to release various growth
factors (TGF-β, IGF-1 and -2, bone morphogenetic proteins [BMPs], bone matrix proteins
[collagen I, osteocalcin, osteopontin]) and thereby counterbalance the reduction of bone
turnover during treatment with bisphosphonates [18-21]. This osteoanabolic effect of
alfacalcidol administered in combination with alendronate has been demonstrated by several
studies [21-24].
Our study was conducted on patients whose bone density was declining despite adequate
treatment with a bisphosphonate and supplementation of calcium plus conventional vitamin
D3 since a year at least. Patients with renal impairment or secondary osteoporosis were not
allowed to participate in the study. Notwithstanding this, the study population included a
substantial number of patients with decreased serum 25-hydroxyvitamin D3 levels (n=38),
accompanied by higher than normal serum PTH level (n=23). This might suggest a potential
relationship between resistance to bisphosphonates and the level of vitamin D3 insufficiency
and the increase of serum PTH. Persistence of secondary hyperparathyroidism reduces BMD
response to alendronate in older women with osteoporosis [9]. The improvement of the
biochemical markers of bone turnover and increasing BMD indicate that patients apparently
resistant to treatment with a bisphosphonate plus supplementation with calcium and
conventional vitamin D3 are more likely to benefit from combination therapy with a
bisphosphonate and alfacalcidol [9]. The enhanced efficacy of alendronate and alfacalcidol in
combination is probably related to the synergism between the considerably different modes of
action of the two components. In particular, the antiresorptive effect of alendronate is
favourably supplemented by the mitigation of osteoclastogenesis [23, 25] and reduction of
serum PTH level, along with the stimulation of osteoblast activity [21], improvement of bone
quality [15,23] and the microarchitecture of trabecular bone by alfacalcidol [23]– which also
enhances muscle strength and thereby activates the ‘mechanostat’ function of the skeleton
[18]. The whole array of these effects might explain the outstanding effect on bones [17-18,
24]. Further elucidation of these mechanisms would require large-scale studies with fracture
endpoints, defined in addition to monitoring changes in bone density.

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Although the lack of a control group may be considered a potential flaw of this study, the
inclusion of untreated patients losing more than 3 per-cent of their bone density each year
would have been ethically questionable at the least.

Conclusion
As shown by the results of this study, patients whose bone density decreases inexorably
despite adequate treatment with alendronate and supplemental calcium and vitamin D3 might
benefit from a combined treatment of alendronate with alfacalcidol. The latter combination
accomplished significant increase of BMD along with improvement of the biochemical
markers of bone turnover, but without any substantial increase in the incidence of adverse
effects.

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Figure legends

Figure 1. The changes of BMD during follow-up. Box and whiskers plots are summarizing
the median, quartiles, and extreme values.
Figure 2. Changes of serum calcium and phosphorus levels, as well as of UCa/creatinine ratio
during the study.
Figure 3. The change of serum alkaline phosphatase activity (U/l), parathormone level
(pg/ml), osteocalcin level (ng/ml ) and of urinary deoxypirydinoline
crosslinks/creatinine (D-Pyr/creatinine, nmol/mmol) by the end of the follow-up.
Figure 4. The ranges of relative changes expressed as percentages of the base values for all
parameters.

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Figure 1. The changes of BMD during follow-up (g/cm²).

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1
Figure 2. Changes of serum calcium and phosphorus levels (mmol/l), as well as of UCa/creatinine
ratio during the study.