Subcutaneous injection of hyaluronidase with recombinant human insulin compared with insulin lispro in type 1 diabetes.

AIMS: Prandial treatment with human regular insulin for diabetes may result in early postprandial hyperglycaemia and late hypoglycaemia due to its slow onset and long duration of action. This study compared injections of recombinant human insulin (rHI) formulated with recombinant human hyaluronidase [rHuPH20] (INSULIN-PH20) to insulin lispro for prandial treatment in subjects with type 1 diabetes (T1D). METHODS: After a 1-month run-in period using twice-daily insulin glargine (or usual basal insulin therapy for pump users) with prandial lispro, 46 subjects with T1D (42 ± 13 years; body mass index: 26 ± 4 kg/m(2); A1c: 6.8 ± 0.5%) were assigned to INSULIN-PH20 or lispro in a random sequence for two consecutive, 12-week periods as the prandial insulin in an intensive treatment regimen. RESULTS: The mean glycaemic excursion for INSULIN-PH20 (0.96 ± 2.00 mmol/l) was comparable (p = 0.322) to lispro (0.80 ± 1.95 mmol/l). The 8-point self-monitored blood glucose profiles were also comparable in the two groups. Good glycaemic control (A1c) was maintained for both treatments at 12 weeks (INSULIN-PH20: 7.0 ± 0.5%; lispro: 6.9 ± 0.6%). Overall rates of hypoglycaemia (≤ 3.9 mmol/l) were 24 events per patient per 4 weeks for INSULIN-PH20 and 22 events for lispro. There were no significant differences in adverse events or immunogenicity between treatments and both treatments were well tolerated. CONCLUSIONS: Unlike commercially available formulations of regular human insulin, a formulation of rHI with rHuPH20 was comparable to lispro for postprandial glucose excursions in a basal-bolus treatment regimen for T1D patients. Glycaemic control, safety and tolerability profiles were comparable for both treatments.

Quality control of DL,CO instruments in global clinical trials.

High inter- and intra-laboratory variability exists for the single-breath diffusing capacity of the lung for carbon monoxide (D(L,CO)) test. To detect small changes in diffusing capacity in multicentre clinical trials, accurate measurements are essential. The present study assessed whether regular D(L,CO) simulator testing maintained or improved instrument accuracy and reduced variability in multicentre trials. The 125 pulmonary function testing laboratories that participated in clinical trials for AIR(R) Inhaled Insulin validated and monitored the accuracy of their D(L,CO) measuring devices using a D(L,CO) simulator, which creates known target values for any device. Devices measuring a simulated D(L,CO) different from target by >3 mL.min-1.mmHg(-1) failed testing and were serviced. Device accuracy was assessed over time and with respect to differences in several variables. Initially, 31 (25%) laboratories had a D(L,CO) device that failed simulator testing. After fixing or replacing devices, 124 (99%) laboratories had passing devices. The percentage of failed tests significantly decreased over time. Differences in geographical region, device type, breath-hold time, temperature and pressure were not associated with meaningful differences in D(L,CO) device accuracy. Regular diffusing capacity of the lung for carbon monoxide simulator testing allows pulmonary function testing laboratories to maintain the accuracy of their diffusing capacity measurements, leading to reduced variability across laboratories in multicentre clinical trials.

Effect of upper respiratory tract infection on AIR inhaled insulin pharmacokinetics and glucodynamics in healthy subjects.

The suitability of employing AIR Inhaled Insulin (AIR Insulin; AIR is a registered trademark of Alkermes) during acute upper respiratory tract infection (URI) has not been determined. Twenty-one healthy, non-diabetic subjects were enrolled in a single-sequence, two-period, euglycemic clamp study. Subjects received a single 12 U-equivalent dose of AIR Insulin before rhinovirus (RV16) inoculation and during symptomatic infection. Spirometry was used to evaluate pulmonary safety. AIR Insulin exposure (the area under the immunoreactive insulin (IRI) concentration vs time curve from time zero until the IRI concentrations returned to the predose baseline value (AUC(0-t'))) and glucodynamic response (total amount of glucose infused (G(tot))) were comparable before and during RV infection (AUC(0-t') 46,300 vs 52,600 pmol min/l, P=0.21; G(tot) 61,800 vs 68,700 mg, P=0.42, respectively). Variability of pharmacokinetic and pharmacodynamic parameters did not change during URI; either did the number or intensity of adverse events. No significant change in forced expiratory volume or forced vital capacity was observed following AIR Insulin administration or during URI. The AIR Insulin system provides similar pharmacokinetic and glucodynamic responses under conditions of an experimentally induced RV infection and is regarded as suitable for use in diabetic patients during URIs.

Efficacy and safety of preprandial human insulin inhalation powder versus injectable insulin in patients with type 1 diabetes.

AIMS/HYPOTHESIS: The efficacy and safety of human insulin inhalation powder (HIIP) plus insulin glargine were compared to subcutaneously injected insulin (SC insulin) plus insulin glargine in patients with type 1 diabetes. METHODS: This was a randomised, open-label crossover study in which one group of patients received preprandial HIIP plus insulin glargine for 12 weeks, followed by the same duration with preprandial SC insulin (lispro or regular) plus insulin glargine. Another group of patients received the reverse treatment sequence. The trial was designed as a non-inferiority comparison of the two treatments for effect on HbA(1c); blood glucose levels were also monitored. Safety assessments included adverse event reporting and hypoglycaemic events. RESULTS: HbA(1c) at endpoint was 7.95+/-0.12% for the HIIP treatment and 8.06+/-0.12% for the SC insulin treatment; mean changes from baseline to endpoint were -0.08 and 0.00%, respectively, (p=NS). The upper limit of the 95% CI of mean difference in HbA(1c) between the two treatments was 0.02%, indicating that HIIP was not inferior relative to SC insulin, as measured against the pre-defined margin of 0.3%. Fasting blood glucose was significantly lower for HIIP treatment (8.09+/-0.33 mmol/l; n=117) than for SC insulin treatment (9.05+/-0.33 mmol/l; n=111) (p=0.01). Safety profiles were comparable between the two treatments. The rate of any hypoglycaemia (least-squares mean adjusted for 30 days+/-SE) was 8.9+/-0.7 and 8.2+/-0.8 for HIIP and SC insulin treatments, respectively, (p=0.29). The rate of nocturnal hypoglycaemia was greater for HIIP (4.2+/-0.4) than for SC insulin (2.7+/-0.4; p<0.001). CONCLUSIONS/INTERPRETATION: HIIP was similar in efficacy to SC insulin for glycaemic control in type 1 diabetes mellitus. The two treatments had comparable safety profiles.

Estimating dose equivalence for new routes of drug administration.

For patient's convenience, dose administration of insulin via oral inhalation is often considered as an alternative to subcutaneous administration. An important statistical problem is to estimate dose equivalence, which is the amount of drug needed to be delivered by inhalation to generate an equivalent pharmacokinetic (PK) response produced by a therapeutic dose of subcutaneous insulin. Because of high intersubject variability, a crossover design clinical trial is typically used where data from both routes of administration are obtained from the same subject. A linear mixed effects model is proposed to describe the relationship between AK response and insulin dose for the two routes of administration. Estimation of dose equivalence in this setting has not been discussed in the statistical literature. Several competing methods for estimating dose equivalence are proposed and contrasted. A formula for calculating an approximate sample size necessary to estimate dose equivalence with a desired precision for the new route of administration is also provided.

Inhaled micronized crystalline human insulin using a dry powder inhaler: dose-response and time-action profiles.

AIM: The aim of this euglycaemic glucose clamp study was to investigate the pharmacokinetics, glucodynamics, safety and tolerability of micronized crystalline human insulin inhalation powder delivered by a Spiros dry powder inhaler system in healthy volunteers. METHODS: Thirteen healthy, non-smoking, male and female volunteers [age 30 +/- 7 years; BMI 23.5 +/- 2.7 kg/m(2); (mean +/- sd)] with normal pulmonary function participated in an open-label, randomised, 6-period crossover trial. Each volunteer received four single doses of inhaled insulin (60, 90, 120, 150 U) on separate occasions. For comparison, each volunteer also received two of three possible doses of subcutaneous (s.c.) injected regular human insulin (8, 14, or 20 U). RESULTS: Serum immunoreactive insulin following inhalation of insulin peaked an average of 60 min earlier compared with s.c. injected insulin (P < 0.0001). Following inhalation, the time to maximum glucose infusion rate occurred an average of 70 min earlier than with s.c. insulin: 187, 129, 161 and 162 min vs. 227, 241 and 241 min (P < 0.0001). The dose-response relationships for serum insulin pharmacokinetics and glucodynamics were linear for both inhaled and s.c. insulin. Relative bioavailability (based on serum insulin levels) ranged from 11.5 to 12.2% for the four doses of inhaled insulin and relative biopotency (based on glucose infusion rates) was 10.0 to 16.5%, respectively. Dosing was well tolerated by all volunteers. CONCLUSION: This study demonstrates that inhalation of human insulin via a dry powder inhaler system provides a promising alternative route for administration of insulin.

Effect of raloxifene on breast cancer cell Ki67 and apoptosis: a double-blind, placebo-controlled, randomized clinical trial in postmenopausal patients.

PURPOSE: Raloxifene is a selective estrogen receptor (ER) modulator approved for prevention and treatment of postmenopausal osteoporosis. This is an exploratory study of raloxifene in primary breast cancer patients. EXPERIMENTAL DESIGN: Postmenopausal women (50-80 years of age), with histological or cytological diagnosis of stage I or II primary breast cancer, were randomly assigned to 14 days of placebo, 60 mg/day raloxifene, or 300 mg twice daily (600 mg/day) of raloxifene. A core biopsy of the primary tumor was obtained before therapy, and a representative sample of the excised tumor was obtained from the operative specimen after treatment. Paired baseline and endpoint biopsies from each patient were analyzed for Ki67, apoptosis, and estrogen and progesterone receptors. Treatment group differences in efficacy measurements were primarily evaluated for baseline-to-endpoint change and percentage change using a one-way ANOVA with treatment as the fixed effect. RESULTS: Of 167 enrolled patients, 143 had evaluable efficacy data. Most breast cancer cases were invasive (98.6%), stage I (76.6%), and ER-positive (83.2%). In patients with ER-positive tumors, Ki67 increased 7% from baseline on placebo and decreased by 21% on 60 mg/day raloxifene (P = 0.015 versus placebo) and by 14% on 600 mg/day raloxifene (P = 0.064 versus placebo). Raloxifene did not affect apoptosis. ER decreased significantly with 60 mg/day or 600 mg/day raloxifene compared with placebo (P < 0.01 for each comparison). Raloxifene had no statistically significant effects on Ki67 among patients with ER-negative tumors. There were no treatment differences in adverse events. CONCLUSION: In this exploratory trial, 60 mg/day raloxifene showed a significant antiproliferative effect in ER-positive breast cancer, demonstrated by the decrease in Ki67, with no effect in ER-negative cancer. This provides support for raloxifene having a breast cancer preventive effect in postmenopausal women.

Continued breast cancer risk reduction in postmenopausal women treated with raloxifene: 4-year results from the MORE trial. Multiple outcomes of raloxifene evaluation.

Raloxifene, a selective estrogen receptor modulator approved for the prevention and treatment of postmenopausal osteoporosis, has shown a significant reduction in breast cancer incidence after 3 years in this placebo-controlled, randomized clinical trial in postmenopausal women with osteoporosis. This article includes results from an additional annual mammogram at 4 years and represents 3,004 additional patient-years of follow-up in this trial. Breast cancers were ascertained through annual screening mammograms and adjudicated by an independent oncology review board. A total of 7,705 women were enrolled in the 4-year trial; 2,576 received placebo, 2,557 raloxifene 60 mg/day, and 2,572 raloxifene 120 mg/day. Women were a mean of 66.5-years old at trial entry, 19 years postmenopause, and osteoporotic (low bone mineral density and/or prevalent vertebral fractures). As of 1 November 1999, 61 invasive breast cancers had been reported and were confirmed by the adjudication board, resulting in a 72% risk reduction with raloxifene (relative risk (RR) 0.28, 95% confidence interval (CI) 0.17, 0.46). These data indicate that 93 osteoporotic women would need to be treated with raloxifene for 4 years to prevent one case of invasive breast cancer. Raloxifene reduced the risk of estrogen receptor-positive invasive breast cancer by 84% (RR 0.16, 95% CI 0.09, 0.30). Raloxifene was generally safe and well-tolerated, however, thromboembolic disease occurred more frequently with raloxifene compared with placebo (p=0.003). We conclude that raloxifene continues to reduce the risk of breast cancer in women with osteoporosis after 4 years of treatment, through prevention of new cancers or suppression of subclinical tumors, or both. Additional randomized clinical trials continue to evaluate this effect in postmenopausal women with osteoporosis, at risk for cardiovascular disease, and at high risk for breast cancer.

Raloxifene: A selective estrogen receptor modulator (SERM) with multiple target system effects.

Selective estrogen receptor modulators (SERMs) exhibit a pharmacologic profile characterized by estrogen agonist activity in some tissues with estrogen antagonist activity in other tissues. These compounds were initially called "antiestrogens," but it was subsequently recognized that this inadequately described their spectrum of activities. The first widely used SERM, tamoxifen, has estrogen antagonist activity in breast tissue but shows estrogen-like activity in other tissues. Raloxifene is another SERM in clinical use, and it was developed to avoid some of the undesirable estrogen agonist actions of other SERMs to improve the drug safety profile. Raloxifene has been introduced for clinical use in treatment and prevention of postmenopausal osteoporosis. This review will explore the preclinical and clinical pharmacology of raloxifene, and compare it to other SERMs currently available for clinical use.

A comparison of the effects of raloxifene and estrogen on bone in postmenopausal women.

Raloxifene HCl, a selective estrogen receptor modulator, has been shown to increase bone mineral density (BMD) and decrease biochemical markers of bone turnover in postmenopausal women without stimulatory effects on the breast and uterus. However, it is not known whether the changes in BMD and bone turnover are associated with changes at the tissue level, nor how changes with raloxifene compare with estrogen. In this randomized, double blind study, we evaluated the effects of raloxifene (Evista, 60 mg/day) or conjugated equine estrogens (CEE; Premarin, 0.625 mg/day) on bone architecture, bone turnover, and BMD. Iliac crest bone biopsies were obtained at baseline and at the end of the study after double tetracycline labeling and were analyzed for standard histomorphometric indexes. Serum and urinary biochemical markers of bone turnover were measured at baseline and at 4, 10, 18, and 24 weeks of treatment. Total body, lumbar spine, and hip BMD were measured at baseline and at the end of the study by dual energy x-ray absorptiometry. Activation frequency and bone formation rate/bone volume were significantly decreased from baseline in the CEE, but not in the raloxifene, group. Bone mineralization did not change in either group. Most markers of bone resorption and formation decreased in both groups, but to a greater degree in the CEE group (P < .05). Total body and lumbar spine BMD increased from baseline in both groups, with a greater increase in the CEE group (P < 0.05). Hip BMD significantly increased from baseline in the raloxifene group, but the change was not different from that in the CEE group. These results suggest that raloxifene reduces bone turnover and increases bone density, although to a lesser extent than CEE. Thus, raloxifene is an alternative to CEE for the prevention and treatment of osteoporosis in postmenopausal women.

Glimepiride, a new once-daily sulfonylurea. A double-blind placebo-controlled study of NIDDM patients. Glimepiride Study Group.

OBJECTIVE: To compare the efficacy and safety of two daily doses of the new sulfonylurea, glimepiride (Amaryl), each as a once-daily dose or in two divided doses, in patients with NIDDM. RESEARCH DESIGN AND METHODS: Of the previously treated NIDDM patients, 416 entered this multicenter randomized double-blind placebo-controlled fixed-dose study. After a 3-week placebo washout, patients received a 14-week course of placebo or glimepiride 8 mg q.d., 4 mg b.i.d., 16 mg q.d., or 8 mg b.i.d. RESULTS: Fasting plasma glucose (FPG) and HbA1c values were similar at baseline in all treatment groups. The placebo group's FPG value increased from 13.0 mmol/l at baseline to 14.5 mmol/l at the last evaluation endpoint (P < or = 0.001). In contrast, FPG values in the four glimepiride groups decreased from a range of 12.4-12.9 mmol/l at baseline to a range of 8.6-9.8 mmol/l at endpoint (P < or = 0.001, within-group change from baseline; P < or = 0.001, between-group change [vs. placebo] from baseline). Two-hour postprandial plasma glucose (PPG) findings were consistent with FPG findings. In the placebo group, the HbA1c value increased from 7.7% at baseline to 9.7% at endpoint (P < or = 0.001), whereas HbA1c values for the glimepiride groups were 7.9-8.1% at baseline and 7.4-7.6% at endpoint (P < or = 0.001, within-group change from baseline; P < or = 0.001, between-group change from baseline). There were no meaningful differences in glycemic variables between daily doses of 8 and 16 mg or between once- and twice-daily dosing. Adverse events and laboratory data demonstrate that glimepiride has a favorable safety profile. CONCLUSIONS: Glimepiride is an effective and well-tolerated oral glucose-lowering agent. The results of this study demonstrate maximum effectiveness can be achieved with 8 mg q.d. of glimepiride in NIDDM subjects.

Self-monitoring of blood glucose in overweight type 2 diabetic patients.

Self-monitoring of blood glucose (SBGM) is widely recommended for both type 1 and type 2 diabetic patients despite the lack of evidence of benefit in glucose control or as an aid in weight loss in type 2 subjects. This study tested the hypothesis that combined use of SMBG and dietary carbohydrate (CHO) counting, using the blood monitoring results to shape dietary CHO quotas, is beneficial in managing type 2 diabetes. Twenty-three overweight (body mass index, BMI 27.5-44 kg/m2) patients aged 40-75 participated in a 28-week behavioral weight control program. Baseline hemoglobin HbA1c ranged between 9.5% and 13.5% (normal range 5.5%-7.7%). Subjects were matched for weight, sex, and HbA1c and assigned to small (4-8 participants) groups which met weekly for 12 weeks and then monthly for 16 weeks. After 8 weeks, the groups were randomized either to continue the behavioral program or to have SMBG and dietary CHO counting. Glucose monitoring was performed 6 times daily (pre- and 2 h postprandially) for the first month, focusing on the meal increment and correlating this to dietary CHO intake. Weight loss was identical in both groups during the year of follow-up. The HbA1c level showed a progressive decline in experimental subjects (P < 0.05), whereas there was no improvement in control subjects.

Do estrogens improve bone mineral density in osteoporotic women over age 65?

A retrospective analysis of our experience with estrogen and fluoride treatment in 91 patients with postmenopausal osteopenia followed for 6-47 months has been performed. Treatment included calcium (1000 mg/day) and either conjugated estrogens (0.625 mg/day) or sodium fluoride (50 mg/day), or both. All patients had at least two serial dual-photon spinal bone mineral density measurements performed 6 months or more apart. Estrogen treatment was associated with increased bone mineral density (5.3%/year), as was fluoride alone (7.5%/year). Estrogen and fluoride together were additive (9.6%/year). In women over age 65 the estrogen effect was just as great (6.9%/year) as in younger women. Estrogen benefit occurred predominantly in the first 18 months of treatment (7.0%/year), after which time changes in bone mineral density were similar to those in untreated controls, who showed stable bone mineral density. We conclude that aggressive estrogen and fluoride treatment tailored to the severity of the individual's postmenopausal osteopenia results in short-term improvement in spinal bone mineral density. These data further support that elderly women respond to estrogen replacement therapy with absolute and relative increments in bone density similar to those in younger women.

Protein folding: assignment of the energetic changes of reversible chemical modifications to the folded or unfolded states.

Reversible chemical modifications of a series of single cysteine-containing variants of T4 lysozyme combined with thermal denaturation studies have been used to study the effects of these modifications on the stability of the protein. This allows dissection of the energetic effects of the modification on both the native and denatured states of this protein. At some sites modifications with various chemical reagents have essentially no effect on the stability of the protein, while at others, substantial changes in stability are observed. For example, chemical modification of cysteine at site 146 by cystamine (+NH3CH2CH2SSCH2-CH2NH3+) to form the mixed disulfide lowers the stability of the protein by about 1.1 kcal/mol. The reduction in the free energy of folding caused by the chemical modification is attributed to the destabilization of native state (0.9 kcal/mol), with only a relatively small effect from stabilization of the denatured state (0.2 kcal/mol). Chemical modifications of T4 lysozyme at site 146 with various chemical reagents show that the stability of the protein is lowered by a positively charged group and is relatively independent of the size of the side chains. This approach allows the investigation of the thermodynamic consequences of the reversible insertion of a wide variety of chemical entities at specific sites in proteins and, most importantly, allows dissection of the contribution of the chemical modifications to both the folded and unfolding states. It can be applied to almost any suitable macromolecular system.

Avoiding reoperation for indeterminate thyroid nodules identified as malignant after surgery.

Thyroid nodules that are indeterminate as carcinoma by needle biopsy before surgery and by study of frozen sections at the time of surgery are occasionally identified to be malignant in later studies. If only a lobectomy has been performed, the advisability of reoperation to remove the remaining thyroid tissue to facilitate radioactive iodine therapy may cause concern. To obviate this difficulty as well as to reduce the occurrence of nodules later in a preserved contralateral thyroid lobe and to provide additional thyroid tissue for study, contralateral subtotal or near total lobectomy has been performed for indeterminate thyroid nodules. The small remnant of remaining thyroid tissue can later be ablated by radioactive iodine if desired. Of 37 patients with indeterminate thyroid nodules, none required reoperation, although the diagnosis of carcinoma was established after surgery for eight patients, three of whom were treated with radioactive iodine.

Bone demineralization, a factor of increasing significance in the management of primary hyperparathyroidism.

The significance of bone demineralization was evaluated for 97 patients treated surgically for primary hyperparathyroidism since 1980. Of 31 patients studied by bone densitometry, 16 showed moderate to severe osteoporosis. In approximately 20% of the total group, bone demineralization, including a bone fracture problem in some, was the dominant or a major indication for operation. Serial bone studies in six patients taken preoperatively or in patients not operated on for primary hyperparathyroidism showed an average loss of bone mineral density of 0.9% per year, whereas in four patients treated surgically serial studies showed an average increase of 9.8% per year. Although estrogen intake reduced serum calcium levels, significant bone demineralization or fractures were present in four patients aged 40 to 59 years and in five patients more than 60 years of age who had taken estrogens for many years. We conclude that in primary hyperparathyroidism, bone demineralization (1) justifies surgical correction in a significant number of patients; (2) should be evaluated, especially in elderly women; (3) is not prevented by estrogen intake, which may instead confuse decision making; (4) is reversed after surgical correction, but suggestions of incomplete reversal emphasize importance of this factor; (5) should be considered in postoperative management; and (6) involves many variables.

Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein.

Twenty-five different temperature-sensitive point mutations at 20 sites in the lysozyme gene of bacteriophage T4 have been identified. All of the mutations alter amino acid side chains that have lower than average crystallographic thermal factors and reduced solvent accessibility in the folded protein. This suggests that the amino acids with well-defined conformations can form specific intramolecular interactions that make relatively large contributions to the thermal stability of the protein. Residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, suggesting that, in general, such amino acids contribute less to protein stability. The pattern of the sites of ts substitutions observed in the folded conformation of T4 lysozyme suggests that severe destabilizing mutations that primarily affect the free energy of the unfolded state are rare. These results indicate that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.