Safety of aromatase inhibitors in the adjuvant setting The third-generation aromatase inhibitors -LRB- AIs -RRB- letrozole , anastrozole , and exemestane are replacing tamoxifen as adjuvant therapy in most postmenopausal women with early breast cancer . Although AIs have demonstrated superior efficacy and better overall safety compared with tamoxifen in randomized controlled trials , they may not provide the cardioprotective effects of tamoxifen , and bone loss may be a concern with their long-term adjuvant use . Patients require regular bone mineral density monitoring , and prophylactic bisphosphonates are being evaluated to determine whether they may protect long-term bone health . AIs decrease the risks of thromboembolic and cerebrovascular events compared with tamoxifen , and the overall rate of cardiovascular events in patients treated with AIs is within the range seen in age-matched , non-breast-cancer populations . AIs are also associated with a lower incidence of endometrial cancer and fewer vaginal bleeding/discharge events than tamoxifen . Compared with tamoxifen , the incidence of hot flashes is lower with anastrozole and letrozole but may be higher with exemestane . Generally , adverse events with AIs are predictable and manageable , whereas tamoxifen may be associated with life-threatening events in a minority of patients . Overall , the benefits of AIs over tamoxifen are achieved without compromising overall quality of life . Introduction Tamoxifen became the standard adjuvant therapy for women with early breast cancer following the first demonstration of efficacy more than 20 years ago -LSB- 1 -RSB- . Administration of tamoxifen for 5 years has been shown to reduce breast cancer recurrence by 41 % and mortality by 34 % in women with hormone-responsive tumors -LSB- 2 -RSB- . Nevertheless , many limitations of tamoxifen have emerged with widespread use . In the landmark National Surgical Adjuvant Breast and Bowel Project B-14 trial , 66 % of tamoxifen-treated patients experienced side effects compared with 58 % of patients given placebo -LSB- 3 -RSB- . Severe , potentially life-threatening events such as thrombosis were more likely to occur in patients aged > 60 years -LSB- 3 -RSB- . Long-term adverse effects associated with 5 years ' adjuvant tamoxifen include venous thromboembolic events , vaginal bleeding , vaginal discharge , ischemic cerebrovascular events , endometrial and uterine cancer , and hysterectomy -LSB- 3 , 4 -RSB- . Experiencing side effects significantly increases the likelihood of patients discontinuing tamoxifen therapy -LRB- odds ratio 4.0 ; 95 % confidence interval -LSB- CI -RSB- 1.1 , 13.9 in women aged ≥ 55 years -RRB- -LSB- 5 -RSB- . Over time , resistance to tamoxifen may develop -LSB- 6 -RSB- , and therapy beyond 5 years is not recommended because neither further disease-free survival nor survival benefit is gained -LSB- 7 -RSB- . The third-generation aromatase inhibitors -LRB- AIs -RRB- letrozole , anastrozole , and exemestane are rapidly replacing tamoxifen as initial adjuvant therapy -LSB- 8 , 9 -RSB- or sequential adjuvant therapy after 2 -- 5 years of tamoxifen -LSB- 10 -- 13 -RSB- . By potently inhibiting the aromatase enzyme , which converts androgens to estrogen -LSB- 14 , 15 -RSB- , AIs achieve almost total suppression of total body aromatization and dramatic reductions in estrogen concentrations in postmenopausal women -LSB- 16 -- 18 -RSB- . AIs are now recommended in international guidelines for the management of breast cancer -LSB- 19 -- 21 -RSB- . In addition , guidance is being developed for the management of common co-morbidities such as osteoporosis in postmenopausal women with hormone-sensitive breast cancer receiving AIs -LSB- 20 , 22 -RSB- . This review examines the safety of AIs and assesses their advantages and disadvantages compared with tamoxifen . It also considers the impact of treatment on co-morbidities commonly encountered in this population . Possible impact of treatment on common co-morbidities Adjuvant therapy should be individualized on the basis of clinical and biologic risk factors -LSB- 21 -RSB- , including the presence of co-morbidities -LSB- 23 -- 26 -RSB- . The most prevalent co-morbidities in the postmenopausal patient population are hypertension , arthritis , heart disease , diabetes , chronic obstructive pulmonary disease , eye problems , anemia , depression , fractures , hearing problems , osteoporosis , Parkinson 's disease , renal failure , and urinary tract problems -LSB- 25 -RSB- . Understanding the long-term effects of aromatase inhibition on bone and cardiovascular health are particularly important to consider because of the potential effects of altering estrogen concentrations . Bone disease Bone health typically may deteriorate as women age , particularly after reaching menopause -LSB- 27 , 28 -RSB- . A decline in estrogen concentrations accelerates postmenopausal bone loss -LSB- 29 -- 31 -RSB- while vitamin D deficiency also increases bone turnover and the risk of fracture -LSB- 32 , 33 -RSB- . It is important to note that bone health is compromised in women with breast cancer compared with the general population -LSB- 34 -RSB- . In the Women 's Health Initiative Observational Study , breast cancer survivors had significantly lower total body bone mineral density -LRB- BMD -RRB- and total hip BMD -LSB- 34 -RSB- and a significantly higher risk of clinical fractures -LSB- 35 -RSB- . Of concern , osteoporosis was undiagnosed in more than three quarters of breast cancer survivors and the reference population -LSB- 34 -RSB- . Multiple factors contribute to the increased risk of osteoporosis and fractures in postmenopausal women with breast cancer -LSB- 34 -RSB- . Furthermore , tumor cells can have a direct effect on bone remodeling -LSB- 36 -RSB- , and breast cancer therapy can lead to cancer treatment-induced bone loss -LRB- CTIBL -RRB- -LSB- 37 -- 39 -RSB- . In a large cohort study , patients with early breast cancer who received anticancer therapy had a 30 % higher risk for osteoporosis/osteopenia -LRB- odds ratio 1.29 ; 95 % CI 1.13 , 1.46 -RRB- -LSB- 38 -RSB- . The study also showed that other factors such as poor health status , history of smoking , and alcohol abuse can contribute to CTIBL . The most serious consequence of CTIBL is an increased risk of fractures -LRB- Fig. 1 -RRB- -LSB- 35 -RSB- , which increase morbidity and healthcare costs -LSB- 40 -RSB- . The presence of bone metastases can contribute to CTIBL and lead to serious complications , including fractures , spinal compression , bone pain , and hypercalcemia of malignancy -LSB- 41 -RSB- . Fig. 1Age-standardized fracture incident rates by survivor status . Standardized rates were calculated using the age distribution of the entire Women 's Health Initiative Observational Study cohort . Excess numbers of fractures per 10,000 person-years are above each set of bars -LSB- 35 -RSB- . © 2005 American Medical Association . Reproduced with permission Aromatase inhibitors and bone disease In a recent study , the bone health of 1,354 patients with breast cancer receiving an AI -LRB- anastrozole , exemestane , or letrozole -RRB- was compared with 11,014 controls -LSB- 39 -RSB- . Treatment with an AI increased the risk of bone loss -LRB- relative risk 1.3 ; 95 % CI 1.1 , 1.6 ; P = 0.01 -RRB- and bone fracture -LRB- relative risk 1.4 ; 95 % CI 1.2 , 1.6 ; P = 0.001 -RRB- . The risks remained significantly higher for AI therapy after adjustment for age and co-morbidities -LSB- 39 -RSB- . An increase in the incidence of arthralgia is noted with all three AIs , when compared with tamoxifen . Anastrozole Howell and colleagues reported fracture rates after a median follow-up of 68 months in the Arimidex , Tamoxifen , Alone or in Combination -LRB- ATAC -RRB- trial -LSB- 42 -RSB- . Fractures were reported in 577 -LRB- 9.3 % -RRB- of the 6,186 patients and were more common with anastrozole than with tamoxifen -LRB- 11 vs. 8 % , respectively ; P < 0.0001 -RRB- . The incidence of hip fractures was 1 % in both groups . The rate of fractures was low at approximately 2 % per year and decreased to baseline levels after completion of 5 years of treatment . The effects of anastrozole and tamoxifen on BMD were assessed in a sub-analysis of 167 patients from the ATAC trial -LSB- 43 -RSB- . Anastrozole-treated patients had significant decreases in lumbar spine BMD -LRB- − 8.1 % ; 95 % CI − 10.1 , − 6.1 ; P < 0.0001 -RRB- and total hip BMD -LRB- − 7.4 % ; 95 % CI − 9.6 , − 5.3 ; P < 0.0001 -RRB- relative to tamoxifen-treated patients , in whom small increases were observed . Bone loss was greatest in the first 2 years of anastrozole treatment , as reported previously -LSB- 44 -RSB- , but the rate of loss appeared to slow down from years 2 to 5 . In the updated analysis after a median follow-up of 68 months , osteopenia or osteoporosis was reported in 11 % of patients receiving anastrozole compared with 7 % receiving tamoxifen -LRB- P < 0.0001 -RRB- -LSB- 42 , 45 -RSB- . Another sub-analysis of the ATAC trial showed that the majority of joint symptoms occur within 24 months of initiating treatment -LSB- 46 -RSB- . After 68 months ' median follow-up , joint symptoms were reported in 35.6 and 29.4 % of patients in the anastrozole and tamoxifen arms , respectively . Most symptoms were mild in intensity , and 46 % were reported as an exacerbation of a pre-existing condition . The incidence of serious joint symptoms was similar for anastrozole and tamoxifen -LRB- 10.6 vs. 10.4 % , respectively -RRB- and only 2.1 and 0.9 % , respectively , discontinued treatment because of joint symptoms . After a median follow-up of 68 months , muscle cramps were less common with anastrozole than tamoxifen -LRB- 4 vs. 8 % , respectively ; P < 0.0001 -RRB- , whereas carpal-tunnel syndrome was more common with anastrozole -LRB- 3 vs. 1 % , respectively ; P < 0.0001 -RRB- -LSB- 42 -RSB- . These updated results from the ATAC trial confirm that AIs are a well-tolerated initial treatment option in terms of bone health -LSB- 43 , 45 , 46 -RSB- . Although anastrozole is associated with BMD loss , no patient with normal bone at baseline became osteoporotic after 5 years of treatment , and the rate of bone loss in the lumbar spine region slowed down in years 2 -- 5 . The ARNO/ABCSG8 trials investigated the efficacy and safety of switching to anastrozole after 2 years of tamoxifen -LSB- 12 -RSB- . Although there were significantly more fractures in patients switching to anastrozole -LRB- 2.1 % -RRB- than in those continuing on tamoxifen -LRB- 1.0 % -RRB- -LSB- 12 -RSB- , the rate was lower than that seen at a similar point in the ATAC trial -LSB- 12 -RSB- . In the Italian Tamoxifen Anastrozole -LRB- ITA -RRB- trial , switching to anastrozole after 2 -- 3 years of tamoxifen was not associated with an increase in fracture rate , although differences may emerge with longer follow-up -LSB- 13 -RSB- . Letrozole In the Breast International Group -LRB- BIG -RRB- 1 -- 98 trial of initial adjuvant therapy , there was a slight yet significant difference in the incidence of fractures -LRB- 5.7 % with letrozole vs. 4.0 % with tamoxifen ; P < 0.001 -RRB- -LSB- 8 -RSB- . The MA .17 trial of extended adjuvant therapy showed that when compared with placebo , letrozole had no significant impact on fractures -LSB- 10 -RSB- . There was a small but significant difference in patient-reported diagnoses of new-onset osteoporosis -LRB- 8 % letrozole vs. 6 % placebo , P = 0.003 -RRB- , and arthralgia and myalgia were significantly more common with letrozole than placebo -LSB- 10 -RSB- . A companion study to MA .17 demonstrated a significant decrease in lumbar spine BMD -LRB- − 5.35 vs. − 0.70 % ; P = 0.008 -RRB- and total hip BMD -LRB- − 3.6 vs. − 0.71 % ; P = 0.044 -RRB- over 2 years in patients treated with letrozole compared with placebo , although no patient went below the threshold for osteoporosis in total hip BMD -LSB- 47 -RSB- . Data from this companion study suggest that women with a BMD score of − 1.0 or greater when starting letrozole after tamoxifen are less vulnerable to enhanced bone resorption and may not require prophylactic bisphosphonate therapy . Exemestane In a model of ovariectomized rats , the steroidal AI exemestane was shown to prevent bone loss , presumably via its androgenic properties -LRB- both exemestane and its metabolite 17-hydro-exemestane demonstrate affinity for the androgen receptor -RRB- -LSB- 48 -RSB- . However , a randomized study to compare the effects of progestins and AIs on bone remodeling markers in patients with metastatic breast cancer found that exemestane increased osteoclast activity -LSB- 49 -RSB- . In the adjuvant treatment setting , a randomized trial involving 147 patients with early breast cancer demonstrated a non-significant effect of exemestane compared with placebo on the annual rate of BMD loss in the lumbar spine -LRB- 2.17 vs. 1.84 % ; P = 0.568 -RRB- and a small but significant effect in the femoral neck -LRB- 2.72 vs. 1.48 % ; P = 0.024 -RRB- -LSB- 50 -RSB- . Of note was the finding that BMD may rapidly improve following AI discontinuation : this trial showed that bone resorption markers returned to or below baseline values , and bone formation markers remained moderately increased within 6 months of stopping exemestane -LSB- 51 -RSB- . In the Intergroup Exemestane Study -LRB- IES -RRB- of exemestane following 2 -- 3 years of tamoxifen , fractures were reported more frequently with exemestane than with tamoxifen after a median follow-up of 30.6 months , although this difference was not statistically significant -LRB- 3.1 vs. 2.3 % ; P = 0.08 -RRB- -LSB- 52 -RSB- . However , the difference in incidence of fractures was statistically significant -LRB- 7.0 % with exemestane vs. 4.9 % with tamoxifen ; P = 0.003 -RRB- after a median follow-up of 55.7 months -LSB- 11 -RSB- . The incidence of osteoporosis was also significantly higher with exemestane than with tamoxifen -LRB- 9.2 vs. 7.2 % , respectively ; P = 0.01 -RRB- . Recent results from a 1-year sub-study revealed that patients on exemestane experienced a significant decrease in hip BMD , while patients on tamoxifen did not -LSB- 53 -RSB- . These results were confirmed by another recent study , which evaluated the effects of exemestane on bone turnover markers and BMD in 70 postmenopausal women -LRB- 62.0 ± 8.9 years -RRB- with early breast cancer who were switched to exemestane after 2 -- 3 years on tamoxifen -LSB- 54 -RSB- . Patients in the exemestane group had a significant decrease in BMD and early parathyroid hormone -LRB- at month 6 -RRB- and an increase in bone alkaline phosphatase -LRB- B-ALP -RRB- and the carboxy-terminal telopeptide of type I collagen after 24 months . These studies suggest that switching postmenopausal women from tamoxifen to exemestane causes a marked increase in bone turnover markers with a consequent reduction in BMD . Arthralgia was also significantly more common with exemestane than with tamoxifen -LRB- 5.4 vs. 3.6 % , P = 0.01 -RRB- in the IES -LSB- 52 -RSB- . A study by Lønning et al. discovered a high prevalence of vitamin D deficiency in postmenopausal women treated with exemestane -LRB- 52 of 59 patients -RRB- or placebo -LRB- 56 of 62 patients -RRB- , and this could be the most important factor causing bone loss in both groups -LSB- 55 -RSB- . Vitamin D substitution is therefore recommended for postmenopausal women , particularly those with breast cancer receiving an AI . The incidence of carpal-tunnel syndrome in the IES was higher in the exemestane arm -LRB- 2.8 % -RRB- than in the tamoxifen arm -LRB- 0.4 % ; P < 0.001 -RRB- -LSB- 11 -RSB- . Comparative studies of aromatase inhibitors A randomized trial -LRB- Letrozole , Exemestane , and Anastrozole Pharmacodynamics -LSB- LEAP -RSB- -RRB- of healthy volunteers demonstrated that letrozole , exemestane , and anastrozole have similar effects on bone biochemical measurements and all result in increases in bone turnover -LSB- 56 -RSB- . There were no statistically significant differences between the AIs in changes from baseline to 24 weeks for B-ALP , serum C-telopeptide crosslinks , and propeptide of type I procollagen . The only difference in the bone remodeling markers was a greater decrease in parathyroid hormone with exemestane than with anastrozole -LRB- P = 0.04 -RRB- . Thus , all AIs seem to have similar effects on bone health . The ATAC bone sub-study results are reassuring for the entire AI class , and women with breast cancer who have normal BMD measurements at the onset of AI treatment may be able to undergo 5 years of therapy without the risk of developing osteoporosis . Patients at risk of clinically relevant BMD loss during treatment should be identified and managed according to evolving clinical guidelines -LSB- 20 , 57 -RSB- . Bisphosphonates In the American Society of Clinical Oncology -LRB- ASCO -RRB- guidelines postmenopausal patients with breast cancer who receive AIs are identified as being at high risk for osteoporosis , and it is recommended that they have baseline BMD evaluation and regular monitoring to guide subsequent therapeutic interventions such as bisphosphonates -LSB- 20 , 58 -RSB- . Preliminary results have been reported from a small number of clinical trials of bisphosphonates in women receiving adjuvant AI therapy . In one trial , premenopausal breast cancer patients receiving goserelin plus anastrozole or goserelin plus tamoxifen were randomly assigned to the bisphosphonate zoledronic acid -LRB- ZA -RRB- -LRB- 4 mg IV every 6 months -RRB- or placebo . After 36 months , it was shown that ZA given every 6 months helped prevent bone loss in these premenopausal patients in both the lumbar spine and hip regardless of endocrine therapy -LSB- 59 -RSB- . Two randomized trials have shown that bisphosphonates may be beneficial in postmenopausal patients at a higher risk of osteoporosis -LSB- 60 , 61 -RSB- . In the Zometa-Femara Adjuvant Synergy Trial -LRB- Z-FAST -RRB- -LRB- North American -RRB- trial , 602 postmenopausal women with hormone-responsive breast cancer starting adjuvant therapy with letrozole were randomized to receive upfront ZA -LRB- 4 mg IV infusion every 6 months -RRB- or delayed ZA when indicated -LRB- either post-baseline T-score decreases < − 2 SD or occurrence of fracture -RRB- -LSB- 60 , 62 -RSB- . Preliminary results after 12 months ' follow-up indicate that initial treatment with ZA may be used to prevent CTIBL , and results at 24 months confirm these initial findings -LSB- 62 , 63 -RSB- although the rate of clinical fractures was not changed . In addition , the small proportion of patients -LRB- 8 % -RRB- requiring ZA in the first year highlights the short-term bone tolerability of letrozole -LSB- 62 -RSB- . Results from the similarly designed ZO-FAST -LRB- European ; N = 1,065 -RRB- trial also support the use of ZA to potentially manage CTIBL in postmenopausal women with early breast cancer receiving adjuvant letrozole -LSB- 61 -RSB- . Lipid metabolism : A cohort study demonstrated that total and low-density lipoprotein -LRB- LDL -RRB- cholesterol concentrations are positively correlated with years since diagnosis of breast cancer -LSB- 64 -RSB- . In addition , during menopause , women experience adverse changes in cardiovascular risk factors , including declines in concentrations of high-density lipoprotein -LRB- HDL -RRB- cholesterol and increases in concentrations of total cholesterol , LDL cholesterol , HDL3 cholesterol , and triglycerides -LSB- 65 , 66 -RSB- . These changes are independent of age and body mass index . Assessing the impact of AIs on lipid profiles is difficult in trials where tamoxifen is the comparator . The selective estrogen-receptor modulators -LRB- SERMs -RRB- such as tamoxifen are known to have lipid-lowering properties -LSB- 67 , 68 -RSB- . What is clear is that the studies comparing AIs with tamoxifen indicate only that the AIs lack the lipid-lowering effects of tamoxifen . Aromatase inhibitors and lipid metabolism Anastrozole In the ATAC trial , the incidence of hypercholesterolemia was higher in patients receiving anastrozole than tamoxifen -LRB- 9 vs. 3 % , respectively ; P < 0.0001 -RRB- -LSB- 42 -RSB- . In the ITA trial , lipid metabolism disorders were reported in 9.3 % of patients treated with anastrozole and 4.0 % receiving tamoxifen -LRB- P = 0.04 -RRB- -LSB- 13 -RSB- . A recent multicenter study in patients with estrogen-receptor positive breast cancer investigated the effects of adjuvant anastrozole and toremifene , a SERM , on serum lipids -LSB- 68 -RSB- . Results showed that only toremifene had a beneficial effect on lipid profile , indicated by a decrease in total cholesterol , LDL cholesterol , triglycerides , and apolipoprotein B , and an increase in HDL cholesterol and apolipoprotein A1 . Changes in total cholesterol , HDL , LDL , and apolipoproteins were significantly different between toremifene and anastrozole at 6 and 12 months -LRB- P < 0.05 -RRB- . Letrozole In the BIG 1 -- 98 trial , according to the protocol , cholesterol concentrations -LRB- fasting or non-fasting -RRB- were collected systematically in the case-report forms every 6 months and even patients with only a single measurement above the upper limit of normal were defined as hypercholesterolemic -LSB- 8 -RSB- . Hypercholesterolemia was reported in 5.4 % of the letrozole arm compared with 1.2 % of the tamoxifen arm in patients with baseline values within normal limits , who then had an increase of 1.5 times the upper limit of normal -LSB- 69 -RSB- . Hypercholesterolemia was typically a single event and in the majority of these patients -LRB- 80 % -RRB- occurred at only grade 1 intensity -LRB- meaning a slight numerical increase above normal , not requiring medications -RRB- . Moreover , the majority of cases were single measurements collected in non-fasting patients . Furthermore , when looking at total serum cholesterol levels , there was a 12 % median decrease from baseline in total cholesterol in the tamoxifen arm after 6 months , consistent with previous reports demonstrating the lipid-lowering effect of tamoxifen -LSB- 67 -RSB- , while in the letrozole group total cholesterol values remained stable -LSB- 8 -RSB- . Hypercholesterolemia was not predefined as an adverse event in the ATAC trial , and lipid concentrations were not routinely assessed -LSB- 42 -RSB- . Exemestane Hypercholesterolemia was not reported in the IES trial of sequential exemestane after tamoxifen -LSB- 11 , 52 -RSB- . Another study examined the longitudinal changes in body composition and lipid profiles in 55 postmenopausal women with early breast cancer switched to exemestane after at least 2 years of tamoxifen treatment -LSB- 70 -RSB- . Fat mass significantly decreased -LRB- P < 0.01 -RRB- while the fat-free mass to fat mass ratio significantly increased -LRB- P < 0.05 -RRB- by month 12 in the exemestane but not in the tamoxifen group . In addition , triglycerides and HDL cholesterol significantly decreased -LRB- P < 0.01 and P < 0.05 , respectively -RRB- in the exemestane group , while LDL cholesterol significantly increased -LRB- P < 0.01 -RRB- at the end of the 1-year study period . Aromatase inhibitors versus placebo When compared with placebo -LRB- the most accurate way to assess the true impact of AIs on serum lipids -RRB- , the final analysis of the MA .17 trial demonstrated the incidence of hypercholesterolemia was 16 % in the letrozole and the placebo arms -LSB- 10 -RSB- . Results from an MA .17 lipid sub-study showed that in 347 postmenopausal women with primary breast cancer treated for up to 36 months , letrozole -LRB- n = 183 -RRB- does not significantly alter lipid profile -LRB- samples drawn under fasting conditions -RRB- compared with placebo -LRB- n = 164 -RRB- -LSB- 71 -RSB- . In a placebo-controlled study involving 147 postmenopausal women with early breast cancer , exemestane had no major effect on lipid profile except for a modest but significant decrease from baseline in HDL cholesterol -LRB- P < 0.001 -RRB- and apolipoprotein A1 -LRB- P = 0.004 -RRB- -LSB- 50 -RSB- . On the basis of these results , it is clear that when compared with placebo , AIs do not have a detrimental effect on lipid profile . However , it should be noted that there have been no placebo-controlled trials of adjuvant anastrozole in women with breast cancer . Comparative studies of aromatase inhibitors The LEAP trial directly compared safety parameters between the steroidal AI exemestane and the non-steroidal AIs anastrozole and letrozole in 90 healthy postmenopausal women -LRB- Table 1 -RRB- -LSB- 72 -RSB- . Initial results from the trial showed that there were no significant differences between anastrozole and letrozole in effects on LDL : HDL ratios , triglyceride concentrations , and non-HDL concentrations . Exemestane was associated with an increase in LDL : HDL ratio -LRB- +17 -RRB- -LRB- P = 0.047 -RRB- compared with anastrozole . There was no median change from baseline in total serum cholesterol for letrozole , a slight increase for anastrozole -LRB- +0.4 -RRB- , and a non-significant decrease for exemestane -LRB- − 3.9 -RRB- -LRB- P = 0.164 vs. anastrozole -RRB- -LSB- 72 -RSB- . Table 1Comparative effects of third-generation aromatase inhibitors on lipids -LSB- 72 -RSB- Percentage change from baselineAnastrozole -LRB- n = 29 -RRB- Letrozole -LRB- n = 29 -RRB- P value vs. anastrozoleExemestane -LRB- n = 32 -RRB- P value vs. anastrozoleTotal cholesterol Week 12 − 2.3 − 3.80.617 − 5.50.262 Week 24 +0.4 − 0.00.900 − 3.90.164 Triglycerides Week 12 − 2.9 +9.60.037 − 7.70.417 Week 24 +0.3 +5.40.550 +2.10.827 Ratio of LDL-C : HDL-C Week 12 − 0.0 − 3.10.486 +8.80.048 Week 24 +4.6 +3.40.847 +17.00.047 Non-HDL-C Week 12 − 2.7 − 4.20.667 − 3.50.820 Week 24 +1.3 +1.20.975 − 0.60.630 Ratio of apo B : apo A1 Week 12 − 1.0 − 3.30.452 +4.40.069 Week 24 +0.0 − 0.80.842 +9.00.023 LDL-C low-density lipoprotein cholesterol , HDL-C high-density lipoprotein , apo B apolipoprotein B , apo A1 apolipoprotein A1 Cardiovascular disease Cardiovascular risk increases substantially and progressively in women aged ≥ 65 years -LSB- 73 -- 77 -RSB- . Isolated systolic hypertension , associated with arterial stiffening , is predominant in middle - and older-aged hypertensives -LSB- 75 -RSB- and predisposes individuals to coronary heart disease , heart failure , stroke , vascular dementia , and chronic kidney disease -LSB- 73 -RSB- . The risk of cardiac disease is also influenced by ethnicity , smoking , obesity , physical inactivity , alcohol abuse , and the presence of co-morbid diseases such as diabetes . In patients with breast cancer the presence of co-morbidities , including cardiovascular disease and diabetes , is associated with a poorer prognosis than when co-morbid disease is absent -LSB- 78 -RSB- and may explain disparities in outcome between different ethnic groups -LSB- 79 -RSB- . There is also evidence that breast cancer is associated with a higher prevalence of hypertension compared with other tumor types -LSB- 80 -RSB- and a significantly increased risk of stroke compared with the general population -LRB- relative risk 1.12 ; 95 % CI 1.07 , 1.17 -RRB- -LSB- 81 -RSB- . Many breast cancer therapies increase the risk of cardiovascular events -LSB- 82 -- 88 -RSB- ; tamoxifen , however , may have some cardio-protective effects -LSB- 89 , 90 -RSB- . Tamoxifen and cardiovascular disease Several studies have demonstrated the potential cardioprotective properties of tamoxifen , including a reduction in hospital admissions due to cardiac disease -LSB- 89 -- 91 -RSB- and decreased mortality from cardiac disease -LSB- 92 -RSB- . In a meta-analysis , tamoxifen was associated with a significantly decreased incidence of myocardial infarction -LRB- relative risk 0.90 -RRB- and death from myocardial infarction -LRB- relative risk 0.62 -RRB- -LSB- 93 -RSB- . This finding is consistent with results from an earlier cohort study -LSB- 94 -RSB- and the Early Breast Cancer Trialists ' Collaborative Group -LRB- EBCTCG -RRB- meta-analysis , which demonstrated decreases in the risk of cardiac death and overall mortality from vascular disease in patients receiving tamoxifen compared with those receiving placebo -LSB- 2 -RSB- . Aromatase inhibitors and cardiovascular disease Assessing the impact of different AIs on cardiovascular disease in postmenopausal women with breast cancer is difficult and inter-trial comparisons are confounded by differences in data collection and end points ; for example , in the BIG 1 -- 98 trial all potential adverse events were predefined in the case-report forms whereas the ATAC trial used non-specific case-report forms to report adverse events -LSB- 8 , 95 -RSB- . Furthermore , comparisons with tamoxifen are complicated by its cardioprotective properties . Placebo-controlled trials thus provide the best source of data to delineate the effects of AIs in a patient population with an inherently elevated risk of cardiac events . Anastrozole The ATAC trial provided data on the cardiovascular effects of anastrozole as initial adjuvant therapy compared with tamoxifen . The incidence of ischemic cardiovascular disease was higher -LRB- but not significantly -RRB- with anastrozole than placebo -LRB- 127/3092 , 4.1 % vs. 104/3094 , 3.4 % ; P = 0.1 -RRB- . The incidence of angina was also higher with anastrozole -LRB- 71/3092 , 2.3 % vs. 51/3094 , 1.6 % ; P = 0.07 -RRB- , while myocardial infarction occurred with similar frequency -LRB- 37/3092 , 1.2 % vs. 34/3094 , 1.1 % ; P = 0.7 -LSB- 42 -RSB- . Hypertension was statistically significantly more common with anastrozole than with tamoxifen -LRB- 13 vs. 11 % , respectively ; P = 0.04 -RRB- -LSB- 42 -RSB- . In the ARNO95 trial vascular events , including hot flashes , ischemic cardiovascular events , deep vein thrombosis , and ischemic cerebrovascular events , occurred in 9.2 % of the anastrozole arm compared with 8.8 % of the tamoxifen arm -LSB- 96 -RSB- . Letrozole The BIG 1 -- 98 trial demonstrated a similar incidence of cardiac events in the letrozole and tamoxifen groups -LRB- 4.1 vs. 3.8 % , respectively ; not significant -RRB- . However , more women in the letrozole group had grade 3 , 4 , or 5 cardiac events -LRB- 2.1 vs. 1.1 % , respectively ; P < 0.001 -RRB- , but these events remain rare -LSB- 8 -RSB- . Of note , a recent update of the monotherapy arms of BIG 1 -- 98 after a longer median follow-up of 51 months showed that the overall incidence of cardiac events was comparable in the two groups -LRB- 134 events -LSB- 5.5 % -RSB- in the letrozole group vs. 122 -LSB- 5.0 % -RSB- in the tamoxifen arm -RRB- , thus confirming the safe cardiac profile of letrozole reported at 26 months -LSB- 97 -RSB- . Exemestane In the IES , there was no significant difference between exemestane and tamoxifen in the incidence of combined cardiovascular disease/thromboembolic events -LRB- 22.1 vs. 20.9 % , respectively ; P = 0.34 -RRB- after a median follow-up of 55.7 months -LSB- 11 -RSB- . The incidence of myocardial infarction was higher with exemestane than with tamoxifen , although the difference between treatment groups was not significant -LRB- 1.3 vs. 0.8 % , respectively ; P = 0.08 -RRB- -LSB- 11 -RSB- . Overall , the rate of cardiovascular events in patients treated with AIs is well within the range seen in age-matched , non-breast-cancer populations ; for example , for women 57 -- 65 years of age , the rates of fatal myocardial infarction and other fatal coronary artery disease are 1.1 and 0.81 per 1,000 patient-years , respectively -LSB- 98 -RSB- . Similar rates were recorded in the UK General Practice Research Database and Swedish MI register -LSB- 99 -RSB- . Currently , there is insufficient information to fully determine the effect of AIs on cardiovascular disease , especially coronary heart disease . Aromatase inhibitors versus placebo Cardiovascular events occurred with similar frequency in the letrozole and placebo arms in the MA .17 trial -LRB- 5.8 vs. 5.6 % , respectively ; P = 0.76 -RRB- -LSB- 10 -RSB- . Similar incidences were reported in the letrozole and placebo arms for stroke/transient ischemic attack -LRB- 0.7 vs. 0.6 % , respectively -RRB- , myocardial infarction -LRB- 0.3 vs. 0.4 % , respectively -RRB- , new or worsening angina -LRB- 1.2 vs. 0.9 % -RRB- , angina requiring coronary artery bypass graft -LRB- 0.2 vs. 0.5 % -RRB- , and thromboembolic events -LRB- 0.4 vs. 0.2 % , respectively -RRB- -LSB- 10 -RSB- . These results clearly indicate that when compared with placebo , AIs do not have a detrimental effect on cardiovascular safety . Gynecologic health The onset of menopause is characterized by numerous adverse events associated with a decline in estrogen concentrations -LSB- 100 -- 102 -RSB- . Early symptoms include abnormal vaginal bleeding , hot flashes , and mood changes , while vaginal dryness and irritation , osteoporosis , and heart disease are late symptoms -LSB- 29 , 103 , 104 -RSB- . Vasomotor symptoms , particularly hot flashes , are common during transition to menopause -LSB- 105 -- 109 -RSB- and may lead to disturbed sleep , depressive symptoms , and significant reductions in quality of life -LSB- 110 -- 115 -RSB- . Cigarette smoking may be associated with increased risk of hot flashes in menopausal women -LSB- 116 -RSB- . Sexual dysfunction is also prevalent in menopausal women and is associated with vaginal atrophy , vaginal/genital dryness , dyspareunia -LRB- pain during sexual intercourse -RRB- , vaginitis , cystitis , and urinary tract infections -LSB- 117 -RSB- . Aromatase inhibitors and gynecologic health Anastrozole In the ATAC trial , the incidence of hot flashes was significantly lower with anastrozole than with tamoxifen -LRB- 36 vs. 41 % ; P < 0.0001 -RRB- -LSB- 9 -RSB- . In the latest analysis , anastrozole was associated with a significantly lower incidence of gynecologic events -LRB- endometrial hyperplasia , endometrial neoplasia , cervical neoplasm , and enlarged uterine fibroids : 3 vs. 10 % with tamoxifen ; P < 0.0001 -RRB- -LSB- 42 -RSB- . A quality-of-life -LRB- QOL -RRB- analysis confirmed that vaginal discharge , vaginal itching/irritation , and vaginal bleeding were less common with anastrozole but found that vaginal dryness , pain during intercourse , and loss of interest in sex were more common -LSB- 118 -RSB- . After 2 years of treatment there was a non-significant trend towards a lower incidence of endometrial abnormalities with anastrozole than tamoxifen -LRB- odds ratio 0.44 ; 95 % CI 0.146 , 1.314 ; P = 0.14 -RRB- -LSB- 119 -RSB- . The latest update of the ATAC trial revealed reduced libido in significantly more patients receiving anastrozole -LRB- 1 % -RRB- than tamoxifen -LRB- < 1 % ; P = 0.0001 -RRB- -LSB- 42 -RSB- . Patients receiving anastrozole also experienced a significantly higher incidence of dyspareunia than those receiving tamoxifen -LRB- 1 vs. < 1 % , respectively ; P = 0.002 -RRB- , whereas urinary incontinence and urinary tract infection were significantly less common among patients receiving anastrozole -LRB- urinary incontinence : 2 vs. 4 % , respectively , P < 0.0001 ; urinary tract infection : 8 vs. 10 % , respectively , P = 0.002 -RRB- . In a randomized study of postmenopausal women in whom abnormal vaginal bleeding and/or asymptomatic endometrial thickening occurred during treatment with tamoxifen , switching to anastrozole was associated with a significant reduction in mean endometrial thickness compared with continuation of tamoxifen -LRB- P < 0.0001 -RRB- -LSB- 120 -RSB- . Significantly fewer anastrozole patients required a repeat hysteroscopy and dilation and curettage compared with those taking tamoxifen -LRB- 4.8 vs. 33.0 % , respectively ; P < 0.0001 -RRB- . Letrozole In the BIG 1 -- 98 trial -LSB- 8 -RSB- , endometrial biopsies were significantly less common in patients receiving letrozole than tamoxifen -LRB- 2.3 vs. 9.1 % , respectively ; P < 0.001 -RRB- , and there was a trend towards fewer invasive endometrial cancers -LRB- 0.1 vs. 0.3 % , respectively ; not significant -RRB- . There was a significantly lower incidence of vaginal bleeding with letrozole than with tamoxifen -LRB- 3.3 vs. 6.6 % , respectively ; P < 0.001 -RRB- , and the incidence of hot flashes was also significantly lower -LRB- 33.5 vs. 38.0 % , respectively ; P < 0.001 -RRB- . In another study in patients intolerant of tamoxifen , switching to letrozole for 6 weeks was associated with a 53.7 % decrease in hot flashes -LRB- hot-flash score 97.0 -- 52.1 ; P = 0.001 -RRB- -LSB- 121 -RSB- . In the MA .17 trial , letrozole was associated with less vaginal bleeding than placebo -LRB- 6 vs. 8 % , respectively ; P = 0.005 -RRB- but a greater incidence of hot flashes -LRB- 58 vs. 54 % , respectively ; P = 0.003 -RRB- -LSB- 10 -RSB- . There was no significant difference in the incidence of vaginal dryness between letrozole and placebo . Exemestane In the IES , there were no significant differences between the exemestane and tamoxifen treatment arms in the incidence of endometrial cancer -LRB- 0.4 vs. 0.7 % , respectively ; P = 0.17 -RRB- -LSB- 11 -RSB- , or the incidence of hot flashes -LRB- 42 vs. 40 % , respectively ; P = 0.28 -RRB- -LSB- 52 -RSB- . Overall , gynecologic symptoms were lower with exemestane than with tamoxifen -LRB- 6 vs. 9 % ; P < 0.001 -RRB- -LSB- 52 -RSB- ; however , vaginal dryness was significantly more common among women taking exemestane than those taking tamoxifen , while vaginal discharge was significantly more common with tamoxifen -LSB- 122 -RSB- . Vaginal bleeding was significantly more common in the tamoxifen arm -LRB- 7.1 % -RRB- than in the exemestane group -LRB- 4.8 % ; P = 0.001 -RRB- -LSB- 11 -RSB- . Other adverse events Secondary cancer The association between tamoxifen and endometrial and uterine cancers is well-established -LSB- 4 -RSB- and is not observed with AIs . However , a safety analysis of the ATAC trial -LSB- 42 -RSB- showed a surprisingly higher incidence of head and neck cancer with anastrozole compared with tamoxifen -LRB- 10/3092 vs. 3/3094 , respectively -RRB- . Similarly , there was an excess of lung cancer -LRB- 25/3092 vs. 16/3094 -RRB- and lung cancer deaths with anastrozole ; however , further analyses are required to confirm these findings . Of note , a higher incidence of secondary cancer was not noted in the IES -LRB- 72 events exemestane vs. 107 tamoxifen -RRB- or in the BIG 1 -- 98 trial -LRB- 69 letrozole vs. 82 tamoxifen -RRB- -LSB- 8 , 11 -RSB- . A meta-analysis showed that tamoxifen is associated with a modest but statistically significant increase in the risk of developing gastrointestinal cancer -LRB- relative risk 1.31 ; 95 % CI 1.01 , 1.69 -RRB- , particularly for postmenopausal women -LRB- relative risk 1.77 -RRB- -LSB- 93 -RSB- . Gastrointestinal health Diarrhea was significantly more common among patients receiving the steroidal AI exemestane than in those taking tamoxifen -LRB- 4.2 vs. 2.2 % , respectively -RRB- -LSB- 123 -RSB- but is not a typical side effect of the non-steroidal AIs letrozole and anastrozole . However , an updated safety analysis of the ATAC trial showed that anastrozole was associated with an increased incidence of diarrhea compared with tamoxifen -LRB- 9 vs. 7 % ; P = 0.02 -RRB- -LSB- 42 -RSB- . Neurologic effects and visual disturbance It has been suggested that endocrine therapy may affect cognitive function in patients with breast cancer -LSB- 124 -RSB- . In a study comparing patients from the ATAC trial with healthy controls , anastrozole was associated with significant impairments in a processing speed task and on a measure of immediate verbal memory -LSB- 125 -RSB- . Another study conducted in healthy , estrogen-treated postmenopausal women treated with testosterone did not reveal any effects of aromatase inhibition on cognition -LSB- 126 -RSB- . The impact of adjuvant AI therapy on cognition and other neurologic processes is clearly an important issue that will require further studies in the future . Neurologic effects reported with exemestane , including dizziness and vertigo -LSB- 127 -RSB- and significantly more visual disturbances compared with tamoxifen -LSB- 52 -RSB- , are not characteristic of non-steroidal AIs . Dry mouth The latest analysis of the ATAC trial demonstrated a significantly greater incidence of dry mouth in patients receiving anastrozole -LRB- 4 % -RRB- compared with tamoxifen -LRB- 2 % ; P = 0.003 -RRB- -LSB- 42 -RSB- . Cosmetic effects Weight gain is common after breast cancer therapy and increases the risk of recurrence , cardiovascular disease , and diabetes -LSB- 64 -RSB- . A study of Japanese patients showed that more women reported weight gain in the anastrozole group than in the tamoxifen group -LRB- 35.8 vs. 12.5 % , respectively ; P ≤ 0.0036 -RRB- -LSB- 128 -RSB- , but no difference was seen among patients from the ATAC trial included in a QOL sub-analysis -LSB- 118 -RSB- . The androgen structure of exemestane may lead to androgenic side effects . Hypertrichosis , hair loss , hoarseness , and acne were reported in about 10 % of patients treated with daily exemestane doses of 200 mg or more in dose-finding studies -LSB- 129 , 130 -RSB- , but have not emerged as a significant issue in phase II or phase III trials with this agent . Anastrozole treatment was associated with a lower incidence of nail disorders -LRB- 2 vs. 3 % ; P = 0.002 -RRB- and fungal infection -LRB- 1 vs. 1 % ; p = 0.01 -RRB- compared with tamoxifen -LSB- 42 -RSB- . Quality of life and patient preference Anastrozole The QOL of patients treated in the ATAC trial was studied during a 5-year follow-up period -LSB- 118 , 131 -RSB- . Anastrozole and tamoxifen had similar overall effects on QOL -LRB- Functional Assessment of Cancer Therapy-Breast -LSB- FACT-B -RSB- trial outcome index plus endocrine sub-scale -RRB- in the first 2 years of treatment -LSB- 118 -RSB- , and an initial worsening of endocrine symptoms gradually improved over time -LSB- 131 -RSB- . The authors concluded that the benefits of anastrozole are achieved without detrimental effects on QOL . However , another study conducted in Japanese patients demonstrated that FACT-G , FACT-B , and FACT-ES scores were significantly better with tamoxifen than with anastrozole -LRB- P = 0.012 , P = 0.010 , and P = 0.015 , respectively -RRB- -LSB- 132 -RSB- . Letrozole The MA .17 and BIG 1 -- 98 trials have demonstrated that adjuvant letrozole is well-tolerated compared with placebo -LSB- 10 -RSB- and better tolerated than tamoxifen -LSB- 8 -RSB- . In another study of postmenopausal women who were experiencing distressing side effects while taking adjuvant tamoxifen and were switched to letrozole , after 6 weeks 66 % of patients preferred to remain on letrozole , 24 % preferred to go back to tamoxifen , and 10 % stopped all therapy -LSB- 121 -RSB- . In the placebo-controlled MA .17 trial , letrozole significantly improved outcomes and did not impair overall QOL -LSB- 133 -RSB- -LRB- Fig. 2 -RRB- . Minor differences seen in some domains -LRB- physical functioning , bodily pain , vitality , vasomotor , and sexual -RRB- were consistent with a minority of patients experiencing changes in QOL compatible with a reduction in estrogen synthesis . A sub-analysis of US subjects in MA .17 demonstrated no significant differences between letrozole and placebo in overall QOL summary scores -LRB- mental and physical -RRB- and five of eight sub-domains of SF-36 -LSB- 134 -RSB- . There were no differences in SF-36 mental and physical QOL scores and MENQOL -LRB- menopause symptom scale -RRB- psychosocial and physical domains -LSB- 134 -RSB- . Fig. 2Mean change score in Short Form 36-item Health Survey . A positive score indicates a favorable change in quality of life . -LRB- A -RRB- Physical component summary ; P = not significant for all time points . -LRB- B -RRB- Mental component summary ; P = not significant for all time points . -LSB- 133 -RSB- . © 2005 American Society of Clinical Oncology . Reproduced with permission Exemestane Results from the IES QOL sub-protocol indicate that switching to exemestane from tamoxifen improves outcome without a significant detrimental impact upon QOL -LSB- 135 -RSB- . At entry , there was a high prevalence of severe endocrine symptoms -LRB- vasomotor complaints and sexual problems -RRB- , and these persisted with exemestane and tamoxifen during the study . No significant differences between groups were seen for any endocrine symptoms apart from vaginal discharge , which was more pronounced with tamoxifen -LRB- P < 0.001 -RRB- . Conclusions Clinical trials show that the third-generation AIs lack the serious risks of thromboembolism and endometrial cancers associated with tamoxifen and are generally well tolerated , with the majority of adverse events occurring at mild to moderate intensity -LSB- 8 -- 11 -RSB- . AIs are associated with a mild to modest increased risk of osteoporosis compared with tamoxifen , and it is therefore essential that patients have regular BMD assessments and be monitored proactively to minimize the risk of clinical fractures -LSB- 20 , 57 -RSB- . The increased risk of fractures with an AI compared with tamoxifen needs to be balanced against the increased risk of endometrial and cerebrovascular/thromboembolic morbidity with tamoxifen -LSB- 136 -RSB- . Of note , the updated ATAC analysis shows that the majority of excess adverse events associated with tamoxifen occurred during the first 2.5 years of treatment ; there were 142 -LRB- 8 % -RRB- fewer predefined adverse events in the anastrozole arm -LSB- 137 -RSB- . Thus , it appears that many excess gynecologic , thromboembolic , and cerebrovascular adverse effects occurring in tamoxifen-treated patients could be avoided if patients were treated initially with an AI -LSB- 136 -RSB- . Although AIs do not have the cholesterol-lowering and potential cardioprotective properties of tamoxifen , they do not significantly worsen total cholesterol concentrations and do not appear to increase cardiovascular risk when compared with placebo . Nevertheless , it is prudent to recommend that all patients at risk of cardiovascular effects are properly monitored and managed , and all breast cancer patients should be routinely monitored for cardiovascular disease . It is difficult to draw meaningful conclusions from comparisons of randomized trials of tamoxifen versus anastrozole , letrozole , or exemestane because of differences in assessing and reporting risk of cardiovascular disease -LSB- 8 , 52 , 95 , 138 -RSB- . Current information is insufficient to determine the effects of AIs on cardiovascular disease and coronary heart disease risk -LSB- 20 -RSB- . Similarly , further follow-up is required to determine the late consequences of AI therapy -LSB- 20 -RSB- . Despite these provisos , ASCO now recommends that optimal adjuvant hormonal therapy for a postmenopausal woman with receptor-positive breast cancer includes an AI as initial therapy or after treatment with tamoxifen . Results from several ongoing trials , including the Femara versus Anastrozole Clinical Evaluation , MA .27 , the National Surgical Adjuvant Breast and Bowel Project , LATER , and MILER , should provide more information on the long-term tolerance and the optimal duration of adjuvant AI therapy and help determine which strategy has the best ratio of efficacy to tolerance . In conclusion , the efficacy benefits of AIs outweigh the risks when AIs are used as adjuvant therapy in postmenopausal women with early breast cancer . Safety , QOL , and patient preference must all be considered in the determination of the optimal strategy for long-term endocrine therapy , bearing in mind that patients may require treatment for 10 years or more . Every patient is unique , and endocrine therapy must be individualized according to clinical , biologic , and patient factors such as lifestyle , the presence of significant co-morbidities , and use of concomitant medications . Tolerability should no longer be an obstacle to effective , long-term endocrine therapy .

B O B I O O O O O O B I O O O B O O O O O O O O O B I O O O O O B I I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

adjuvant therapy
aromatase inhibitors
early breast cancer
letrozole
safety


Solute transport in orthorhombic lysozyme crystals : a molecular simulation study Long-time equilibrium molecular dynamics simulations were performed to study the passage of a substrate , l-arabinose , through nanopores of orthorhombic hen egg white lysozyme crystals . Cross-linked protein crystals -LRB- CLPC -RRB- , as novel biological nanoporous media , consist of an extensive regular matrix of chiral solvent-filled nanopores via which ions and solutes , e.g. sugars and amino acids , travel in and out . We studied the diffusive motion of arabinose inside protein channels . The computed diffusion coefficients within the crystal were orders of magnitudes lower relative to the diffusion coefficient of the solute in water . This study is valuable for understanding the nature of solute -- protein interactions and transport phenomena in CLPCs and provides an understanding of biocatalytic and bioseparation processes using CLPC . Introduction Protein crystals are conventionally thought as important elements in structure determination of biomolecules as well as in protein purification -LRB- Johnson and Philips 1965 ; Blundell et al. 2003 ; Haring and Schnrier 1991 -RRB- . They contain pores that range from approx. 0.3 nm to 10 nm and occupy 25 -- 75 % of the crystal volume -LRB- Bishop and Richards 1968 -RRB- . Their porosity is comparable to that of inorganic porous catalysts and sorbents , such as zeolites and silica-gel -LRB- Bishop and Richards 1968 ; Vilenchik et al. 1998 ; Margolin and Navia 2001 -RRB- . More robust cross-linked protein crystals -LRB- CLPC -RRB- have been applied successfully as extremely stable biocatalysts -LRB- Vilenchik et al. 1998 -RRB- and as selective -LRB- chiral -RRB- separation media -LRB- Margolin and Navia 2001 -RRB- . The complex crystal structure of the protein contains many functional active sites . Moreover , it resists environmental degradation once it is cross-linked -LRB- Vilenchik et al. 1998 ; Margolin and Navia 2001 -RRB- . Understanding the nature of transport of solutes in CLEC is relevant to many biotechnological processes -LRB- Margolin and Navia 2001 ; Margolin and Vilenchik 1996 ; Margolin et al. 2000 ; Vuolanto et al. 2002 , 2004 ; Jokela et al. 2002 -RRB- . Properties of intracrystalline water molecules and ions , and their transport through the crystal , are essential to many of these applications . Apart from their practical use , protein crystals also provide a unique model to study transport phenomena in protein channels in cell membranes since the structure of water-filled pores in the crystals is determined to atomic resolution by X-ray diffraction -LRB- Morozova et al. 1996 -RRB- . There are effects of both the solute on protein and protein on the solute -LRB- Nagendra et al. 1998 -RRB- . The latter effect is more pronounced in protein crystals , where proteins make up the lining of small pores and water and ions affect the passage of substances through such pores -LRB- Nagendra et al. 1998 ; Eisenberg and Kauzmann 1969 -RRB- . Several studies have focused on the experimental determination of the solute and water transport in protein crystals -LRB- Bishop and Richards 1968 ; Morozov et al 1995 ; Velev et al. 2000 ; Cvetkovic et al. 2005a , b ; O'Hara et al. 1995 -RRB- . Recent studies on diffusion in protein crystals still leave open questions concerning the mobility of solutes and protein -- solute interactions near proteins that constitute the pore walls -LRB- Cvetkovic et al. 2005a , b -RRB- . In addition to advanced experimental techniques , versatile computational tools are generally needed to correlate reactivity of the protein and transport of solute with nanoporosity of the enzyme crystals at atomistic level -LRB- Malek et al. 2004 , 2005 -RRB- . Molecular dynamics simulations with explicit representation of molecules and ions should , in principle , provide realistic information about the diffusive motion of water , individual solute molecules and ions at atomic resolution . These simulations , however , are only practical at longer time and length scales . Due to heterogeneity of the protein surface , it is necessary to treat transport of molecules at the protein -- water interface as a local property -LRB- Bon et al. 1999 -RRB- . There have been a number of such studies to determine how the water molecules on the protein surface are perturbed from the bulk -LRB- Bizzari and Cannistraro 1996 ; Bizzari et al. 1996 -RRB- . To our knowledge , there have been no such studies on dynamical motion of small solute molecules in a protein crystal . The mobility of small dipolar molecules , such as sugars and amino acids , in the vicinity and far from the protein surface in a confined biological channel , exhibits different dynamical and structural behavior from that in a free protein in solution -LRB- Velev et al. 2000 ; Cvetkovic et al. 2005a , b ; O'Hara et al. 1995 ; Malek et al. 2004 , 2005 -RRB- . In the crystalline form , proteins are in a highly ordered three-dimensional structure , where the protein molecules strongly bind to each other with specific intermolecular interactions . Protein crystals are particularly interesting materials for chiral separations -LRB- Margolin and Navia 2001 ; Margolin and Vilenchik 1996 ; Margolin et al. 2000 ; Vuolanto et al. 2002 , 2004 ; Jokela et al. 2002 -RRB- . The crystal framework poses an asymmetric environment and is made of l-amino acids . Enzymes in crystals have also specific binding sites for the substrates and cofactors . In addition , protein crystals are nanoporous materials . Based on these properties , many CLPCs have recently been used in chiral separation of racemic mixtures -LRB- Vuolanto et al. 2004 ; Jokela et al. 2002 -RRB- . These novel insights are valuable for biotechnological applications to devise highly selective biocatalysts , biosensors and bioseparators . Here , we use orthorhombic lysozyme as a simple model enzyme to study diffusion of a chiral substrate through pore network of the enzyme crystal . Its well-known and extremely stable molecular structure makes it a good choice for computational studies . Moreover , lysozyme belongs to a large class of enzymes known as glycosidases . These are extremely efficient glycosidic hydrolysis catalysts . Biocatalysis inside the protein lattice has advanced features of product separation and catalyst recovery . Once the catalytic reaction occurs in lysozyme crystals , understanding the transport of products -LRB- sugars -RRB- through the lattice becomes an important issue . In short , the simple and well-known structure of lysozyme as model protein , the catalytic activity of lysozyme for hydrolyzing the glycosidic bond , the practical potential of protein crystals as biochemical porous media for chiral separation and as a model for biological channels , in general , were our first motivations for choosing a simulation model based on transport of sugar substrate into orthorhombic lysozyme lattice . Here we provide , for the first time , long-time MD simulations of diffusion of l-arabinose in orthorhombic hen egg white lysozyme crystals . Fundamental questions are ; do solute molecules move by translation and/or rotation ? How solute -- protein interactions influence transport of a chiral solute through the protein crystal ? What is the detailed nature of the diffusive motion of solute molecules in a lysozyme crystal and what are the length scales and the time scales -LRB- dynamics -RRB- of all those events . Computational methodology We use MD simulations to examine diffusion of l-arabinose -LRB- Ara -RRB- in a fully hydrated orthorhombic lysozyme crystal . Ara is an aldopentose -LRB- Fig. 1 -RRB- that has been extensively used as a substrate to probe diffusion properties of protein channels -LRB- Nikaido and Rosenberg 1981 -RRB- . Lysozyme consists of 129 amino acids with 1001 non-hydrogen atoms . The crystal structures of lysozyme , entry 1AKI -LRB- Artymiuk et al. 1982 -RRB- , is taken from the Brookhaven Protein Database and used as a starting point . Hydrogen atoms attached to aliphatic carbon atoms are incorporated with the latter , but the remaining 342 hydrogen atoms are treated explicitly , leading to 1,343 -LRB- pseudo - -RRB- atoms in total . The simulations are done at pH 7 . The amino acids Glu and Asp are taken to be deprotonated while Lys , Arg and His residues are protonated -LRB- Artymiuk et al. 1982 ; Stocker et al. 2000 -RRB- . This leads to +8 electron charges per protein molecule . Chloride ions are then added for electroneutrality . In an orthorhombic crystal , four protein molecules related by the crystallographic symmetry P212121 are placed in the unit cell with a = 5.9062 nm , b = 6.8451 nm , and c = 3.0517 nm . Repeating the unit cell along crystallographic axes generates the pore network -LRB- Fig. 2 -RRB- . Channels and cavities within the orthorhombic lysozyme lattices are determined by using a procedure explained elsewhere -LRB- Malek et al. 2004 -RRB- , based on HOLE and CHANNEL algorithms -LRB- Smart et al. 1996 ; Kisljuk et al. 1994 -RRB- . The radius of a pore is determined at any given distance along the pore axis by calculating the maximum size for a spherical probe to still fit in the pore without overlap with the van der Waals radii of atoms in the pore wall -LRB- Kisljuk et al. 1994 -RRB- . Fig. 1Ball-and-stick model of l-arabinose . CHn groups are represented by united atomsFig . 2Computer generated images of an orthorhombic lysozyme lattice , visualized along z - crystallographic axis . The solvent channels are shown in grey . A single unit cell is labelled by square and its sphere representation is shown in -LRB- b -RRB- . Red lines represent water molecules . The surface representation in -LRB- a -RRB- was computed from the electron density calculations using the crystal structure data -LRB- 1AKI -RRB- available in the Brookhaven protein data bank . Hydrophilic , hydrophobic and polar regions are shown in blue , red and green respectively Simulations are carried out using a fully atomistic 1 × 1 × 5 lysozyme lattice -LRB- a = 5.9062 nm , b = 6.8451 nm , and c = 15.2585 nm -RRB- . The single unit cell in Fig. 2b is repeated five times along the z-axis , so that a long pore is constructed . We start from an initial configuration by random placing arabinose inside the lysozyme channel . The system contains 20 lysozyme molecules , 21 arabinoses , 6,239 crystallographic and non-crystallographic water molecules and 160 chloride ions , leading to 46,031 atoms in total . After that the system was equilibrated for 500 ps using harmonic position restraints -LRB- 1000 kJ mol − 1 nm − 2 -RRB- . Production runs were performed for another 26 ns , the last 25 ns of which was used for the analysis . Simulations are performed using GROMOS96 force field -LRB- van Gunsteren et al. 1990 , 1996 -RRB- . In our force field , interactions between atoms are divided into non-bonded interactions , between any pair of atoms that are within a given cut-off radius , and bonded interactions between atoms connected by chemical bonds . In case of the non-bonded interactions -LRB- electrostatic and van der Waals -RRB- , a partial charge and parameters for repulsion and attraction are assigned to each atom . The bonded interaction consists of bond , angle and dihedral terms . Bond and angle bending are given by simple harmonic potentials . The torsional rotational potential for the dihedral angle is a periodic function with a 3-fold barrier . The partial charges and interaction parameters for all species are taken from GROMOS96 database -LRB- van Gunsteren et al. 1990 -RRB- . In our MD simulation , a cut-off of 1.4 nm is used for Van der Waals interactions , while a cut-off of 1 nm and PME with a grid spacing of 0.12 nm and fourth order interpolation are used for electrostatics interactions -LRB- Morozova et al. 1996 -RRB- . During the simulations , the potential energy and the total energy are monitored in order to check if the system is in equilibrium . MD simulations are performed in a canonical -LRB- NVT -RRB- ensemble at 300 K . The temperature -LRB- 300 K -RRB- is controlled by the Berendsen weak-coupling algorithm , separately for protein , Ara and solvent plus ions with a time constant of 0.1 ps . During the simulations , polar hydrogen atoms are treated as dummy atoms with an increased mass of 4 Da . This allowed the integration time step to be 5 fs . Simulations are done with the GROMACS package -LRB- Lindahl et al. 2001 ; Berendsen et al. 1995 ; http://www.gromacs.org -RRB- . Visualization is done by using the VMD v1 .8.3 -LRB- Humphrey et al. 1996 -RRB- commercial package . Results and discussions Figure 2a shows an instantaneous configuration of the atomic model of the orthorhombic lysozyme lattice , constructed of 3 × 3 × 3 unit cells , whereas a single unit cell is framed in Fig. 2b . In each unit cell , there is a main pore lies along the z-axis and there are no remarkable pores in the other directions -LRB- Malek et al. 2004 ; Geremia et al. 2005 ; Falkner et al. 2005 -RRB- . Figure 3a visualizes part of the main pores along the z-axis . The average pore radius is about 0.88 ± 0.02 nm . Figure 3b shows the pore radius as a function of the pore axis . The profiles show that there are constricted zones inside each pore . The pore radius slowly decreases from over 0.95 nm to slightly less than 0.82 nm at its narrowest point . The charged residues Lys1 , Lys13 , Lys33 , Lys96 , Lys97 , Lys116 , Arg14 , Arg21 , Arg114 , Asp87 , and Asp119 belonging to the lysozyme molecules lie in the pore space . The motion of these residues may affect the pore shape and pore size during the transport of Ara . Our analysis shows that the effect of the protein fluctuations on pore shape is negligible , particularly for solutes of sizes much smaller than the pore diameter , such as Ara . Fig. 3 -LRB- a -RRB- Visualized pore along z-axis in a 1 × 1 × 5 lysozyme lattice . -LRB- b -RRB- Pore radius profile along the pore axis The averaged root mean square fluctuation -LRB- RMSF -RRB- of C-α of each residue , calculated from the distance fluctuation matrix of a 25 ns trajectory in crystalline orthorhombic lysozyme is shown in Fig. 4a . The root mean square fluctuations from the X-ray structure are averaged over 20 proteins . The fluctuation results -LRB- Fig. 4a -RRB- are consistent with previous works on fluctuations and correlations in lysozyme crystals -LRB- Héry et al. 1997 , 1998 -RRB- . The largest fluctuations are for the α-helix loops . The β-strand residues show a low mobility with values on the order of 0.1 nm . β-Strands exhibit a high degree of rigidity with moderate correlations , while only parts of α-helices are rigid or correlated -LRB- Héry et al. 1998 -RRB- . Although the protein atoms fluctuate slightly during the simulation , these are too small to cause significant changes in pore size or in diffusivity . Figure 4b shows that the RMSF patterns are similar for all twenty lysozyme molecules . The residues Gly117 , Gly71 and Asp101 show fluctuations above 0.10 nm . Among these , Gly117 displays the maximum fluctuations ranging from 0.15 nm to 0.25 nm . Most of the charged residues accessible from the pore space show fluctuations around 0.05 nm . The latter confirms that the pore shape and pore size are not affected by protein fluctuations during 25 ns MD simulation . Fig. 4 -LRB- a -RRB- A typical root mean square fluctuation of the C-α atoms of each residue of one of the 20 lysozyme molecules in the simulation box with respect to their average position . -LRB- b -RRB- RMSD of each resides of all lysozyme molecules . The curves are on the same scale . The full lines are shifted by 0.2 nm in the vertical direction In order to study the motion of solutes inside orthorhombic lysozyme crystals , we take into account that proteins affect the dynamics of solute molecules . MD simulations provide information to the dynamic picture of the solute motion in the vicinity of protein molecules . The self-diffusion coefficient D , which has been widely used in both spectroscopic experiments and MD simulations , is a suitable parameter characterizing the dynamical behavior of solute in the water filled protein channels in protein crystals . The diffusion coefficient D , is related to the slope of the mean square displacement -LRB- MSD -RRB- of water molecules by the Einstein relation , which in d dimension is : where is the MSD of solute molecules during the time Δt , averaged over the ensemble of molecules in a d dimensional space , from the moment t0 that their motion is equilibrated in the pore space . Use of the Einstein equation for the determination of D requires a linear dependence of the MSD with time . For the determination of the solute diffusivity , the MSD -- t linear relation is usually fulfilled on time scales longer than a few to hundred nanoseconds -LRB- Bizzari and Cannistraro 1996 -RRB- . In order to study diffusion of a solute -LRB- Ara -RRB- inside pore space of an orthorhombic lysozyme crystal , we have performed extensive MD simulations of Ara transport in a fully hydrated lysozyme crystal . Protein atoms are subject to move according to the equation of motions . Ara molecules are randomly incorporated inside the main pore along z-axis -LRB- Fig. 3a -RRB- . The dynamical motion of solute molecules was sampled each 10 ps during 25 ns simulation . Figure 5a shows the displacement of nine Ara molecules along the z-axis as a function of time . The location of the solute molecules is sufficiently randomized during the simulation time , so that the diffusion properties do not depend on the arbitrary initial placement of the Ara molecules . The motion of Ara molecules displays many jumps , with little motion between jumps . A few Ara molecules travel within the pore space with displacement in the order of 5 -- 10 nm , while a few others remain around the same axial -LRB- z -RRB- position in the pore region , moving rather in xy plane . Overall , solute molecules move freely , some travel all the way within the pore network , a few remain in the pore and some go deep into the pore and return after some time . At some points along the trajectory , a group of two or three Ara molecules establish hydrogen bonding and perform a collective motion as a united body . The latter is particularly observed in longer simulation times thereby bringing an artefact in to the diffusion analysis of single Ara molecules . To calculate the self-diffusion -LRB- D -RRB- of solute molecules in the pore , the Einstein relationship -LRB- Eq . 1 -RRB- is used . Fig. 5 -LRB- a -RRB- The displacement of nine arabinose molecules along the z-axis through the pore region of lysozyme crystal shown in Fig. 2a . Different colours represent different Ara molecules . -LRB- b -RRB- Mean square displacement -LRB- MSD -RRB- of Ara molecules versus time Figure 4b shows Mean square displacement values -LRB- MSD -RRB- values versus time for Ara molecules diffusing in the pore network of lysozyme crystal . Only trajectories of individual Ara molecules are included in calculation of MSD and therefore the collective motion of solute molecules is not included in Fig. 5b . Overall , the log-log behavior of MSD vs. time -LRB- for 5 ns < t < 20 -RRB- is linear with a slope close to one . This shows that in fully hydrated pores of the lattice , the Einstein relation , Eq . 1 , can describe the diffusion of Ara . The diffusion coefficient of Ara in pores along z-axis is calculated from the intercept of the log -- log plot , that is -LRB- 8.51 ± 0.064 -RRB- × 10 − 13 m2 s − 1 . Recently , we have performed combined dynamic Monte-Carlo and Brownian dynamic -LRB- MC -- BD -RRB- simulations to study electrostatic and steric confinement effects on the mobility of spherical probes in orthorhombic and tetragonal lysosyme crystals -LRB- Malek et al. 2004 -RRB- . The diffusion coefficient of a probe of size comparable to that of the hydrated Ara -LRB- 0.86 ± 0.01 nm -RRB- , calculated by MC -- BD simulations is -LRB- 8.85 ± 0.05 -RRB- × 10 − 13 m2 s − 1 , which is in good agreement with the MD prediction . This indicates that size exclusion is the main reason for uncharged solute transport in orthorhombic lysozyme crystals . The diffusion of solutes within lysozyme crystals has also been investigated using different experimental techniques -LRB- Velev et al. 2000 ; Cvetkovic et al. 2005a , b ; O'Hara et al. 1995 -RRB- . Velev et al. -LRB- 2000 -RRB- studied the diffusion of surfactants in lysozyme crystals using fluorescent probes by means of quantitative fluorescence microscopy . The diffusion coefficients obtained range from 2 to 30 × 10 − 14 m2 s − 1 . By using 3-D laser scanning confocal microscopy visualizations , Cvetkovic et al. -LRB- 2005a , b -RRB- investigated detailed diffusion coefficients of some solutes , fluorescein and rhodamine B , into tetragonal , orthorhombic , and triclinic lysozyme crystals . The results showed that the transport of solute molecules depends on the chemical nature of solutes -LRB- e.g. , hydrophobic vs. hydrophilic -RRB- , charge and size of solutes and pore network within protein crystal . The diffusion of fluorescein , with an average diameter of 0.69 ± 0.02 nm , in orthorhombic lysozyme crystals was highly anisotropic and the diffusion coefficient was calculated as approx. 7.0 ± 0.5 × 10 − 13 m2 s − 1 . The diffusion coefficient of Ara calculated from our MD simulation is in reasonable agreement to these experiments . The diffusion rate is 4 -- 5 orders of magnitude slower than that in free water -LRB- ∼ 7.73 × 10 − 10 m2 s − 1 at 298 K -RRB- -LRB- Nikaido and Rosenberg 1981 ; Mogi et al. 2007 -RRB- . The deviation is most likely as a result of single-point interactions between solute and side chains of the XI , which are usually characterized by hydrogen bindings and dipole-dipole interactions . Our preliminary analysis based on hydrogen bonds , number of contacts and minimum distance between Ara molecules and residues on the pore wall showed that the solute molecules spend considerable time in the vicinity of active site residues Lys96 , Lys97 , Lys116 , Arg14 , Arg21 , and Asp87 . This indicates that the orientation and translational-rotational motion of Ara play an important role in its transport through channels in orthorhombic lysozyme crystals . These results suggest that in lysozyme lattice , and in contrast to the free lysozyme in solution , the interactions between dipolar solute and protein are mostly promoted by a large electric field on the residues far from the active site cleft -LRB- which are Asp52 , Asp53 , Glu35 , and Asp99 -RRB- . Many of the latter charged amino acids that act as adsorption/desorption sites for the solute are , in fact , buried , while the residues Lys96 , Lys97 , Lys116 , Arg14 , Arg21 , and Asp87 are more easily accessible . Considerable experimental and computational evidence supports several aspects of the mechanism that has been proposed for the catalytic activity of the lysozyme in solution -LRB- Dao-Pin et al. 1989 ; Bottoni et al. 2005 -RRB- . In a manner similar to that proposed for free lysozyme , our simulation results can be validated based on finding the most favorable interaction of the substrate molecules in the mixture with the catalytic active centers of the enzyme molecules in lattice . Such qualitative agreement with experimental data corroborates predictive capabilities of our model . Moreover , our study highlights the importance of the protein -- solute interactions on the transport in protein crystals . Despite its simplicity and obvious limitations , this computational study provides insight into the main features of solute transport in protein crystals . Our study allows relating transport properties of the nanopores in protein crystals to solvent and ion motion as well as to protein fluctuations . Although our findings are in good qualitative and quantitative agreement with existing experimental data , more experimental studies are still needed by which we can compare our data directly . These results in combination with experimental information provide vital insights for understanding biocatalytic and chiral separation processes in CLPC . Conclusion In the present study , we performed MD simulations to study the motion of an aldopentose molecule -LRB- l-arabinose -RRB- in nanopores of a fully hydrated orthorhombic lysozyme crystal . The electrostatic and steric interaction inside the crystal channel significantly influences the diffusion of solute . No clear adsorption site in the crystal was detected in the simulation , most likely due to the low solute concentration . However , our analysis show that the solute molecules spend considerable time in the vicinity of active site residues Lys96 , Lys97 , Lys116 , Arg14 , Arg21 , and Asp87 . According to our results , the average mobility of solute molecules in lysozyme crystal channels is reduced orders of magnitude compared to that in free water . This is why crystalline proteins are easily accessible to solute molecules that are smaller than the necks of the crystal channels . The results are of practical interest to evaluate the time necessary to impregnate the crystals with ligands , water and solutes and provide valuable insights of solute -- protein interactions during solute transport in nanopores of protein crystals .

O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

diffusion
lysozyme
molecular dynamics
protein crystal