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Ultra rapid lispro (URLi) is a novel insulin lispro formulation developed to more closely match physiological insulin secretion and improve postprandial glucose control. This study compared the pharmacokinetic profile and glucodynamic response of URLi when administered subcutaneously into the abdomen, upper arm, or thigh. An intravenous (IV) bolus administration was included to determine the absolute bioavailability at each injection site.In this Phase I, randomized, open-label, 4-period, crossover study, healthy subjects received a single dose of 15 U URLi subcutaneously into the abdomen, upper arm, or thigh, or by intravenous injection. Serum insulin lispro concentrations and glucodynamic response during a 10-hour euglycemic clamp procedure were assessed after URLi administration.Total insulin lispro exposure was similar for the abdomen, upper arm, and thigh, and absolute bioavailability was ∼65% at each subcutaneous (SC) injection site. Total and peak insulin action were similar across these SC injection sites. The onset of appearance was1 minute, and the time to early half-maximal drug concentration occurred at ∼10 minutes across these three SC injection sites. Onset of insulin action occurred at ∼22 minutes, and the early insulin action (for the first hour) was also similar across these SC injection sites. URLi was well tolerated after single SC injections and IV bolus administration.The pharmacokinetic and glucodynamic profiles of URLi were similar after a single SC dose into the abdomen, upper arm, or thigh. The rate of insulin lispro absorption and early insulin action were maintained regardless of the SC injection site. The current study supports SC injection of URLi into the abdomen, upper arm, and thigh.gov identifier: NCT03232983.

Typically, therapeutic proteins (TPs) have a low risk for eliciting meaningful drug interactions (DIs). However, there are select instances where TP drug interactions (TP-DIs) of clinical concern can occur. This white paper discusses the various types of TP-DIs involving mechanisms such as changes in disease state, target-mediated drug disposition (TMDD), neonatal Fc receptor (FcRn), or anti-drug antibodies (ADAs) formation. The nature of TP drug interaction being investigated should determine whether the examination is conducted as a standalone TP-DI study in healthy subjects, in patients, or assessed via population pharmacokinetic (PopPK) analysis. DIs involving antibody-drug conjugates are discussed briefly but the primary focus here will be DIs involving cytokine modulation. Cytokine modulation can occur directly by certain TPs, or indirectly due to moderate-to-severe inflammation, infection, or injury. Disease states that have been shown to result in indirect disease-DIs that are clinically meaningful have been listed (i.e., typically a 2-fold change in the systemic exposure of a co-administered sensitive CYP substrate drug). Type of disease and severity of inflammation should be the primary drivers for risk assessment for disease-DIs. While more clinical inflammatory marker data needs to be collected, the use of two or more clinical inflammatory markers (such as c-reactive protein, albumin, or interleukin-6) may help broadly categorize whether the predicted magnitude of inflammatory disease-drug interaction risk is negligible, weak, or moderate-to-strong. Based on current knowledge, clinical DI studies are not necessary for all TPs, and should no longer be conducted in certain disease patient populations such as psoriasis, which do not have sufficient systemic inflammation to cause a meaningful indirect disease-DI.

Introduction Ultra rapid lispro (URLi) is a novel insulin lispro formulation that was developed to more closely match physiological insulin secretion. The aims of this study were to demonstrate the bioequivalence (BE) of a concentrated formulation (U200) of URLi to the U100 formulation of URLi after subcutaneous (SC) administration and to evaluate the glucodynamics (GD) of these formulations. Methods This phase 1, randomized, two‐sequence, four‐period, double-blind, replicate crossover study was conducted in 68 healthy subjects. At each dosing visit, subjects received a 15-U SC dose of either U100 URLi or U200 URLi followed by a 10-h euglycemic clamp procedure. Serum insulin lispro and blood glucose concentrations were measured, and the glucose infusion rate was continuously adjusted during the clamp to maintain the target blood glucose. Results Bioequivalence of U200 URLi relative to U100 URLi was demonstrated. The 90% confidence intervals (CIs) of the ratios of geometric least squares (LS) means for the maximum insulin concentration and total exposure were within the BE limits of 0.80–1.25. Additionally, the 90% CIs for the ratios of geometric LS means for maximum glucose infusion rate and total glucose infused were within the BE limits. The early 50% tmax occurred at approximately the same time for the U100 and U200 URLi formulations, and the insulin exposure within the first 15 min was similar for both formulations. The tolerability of the two URLi formulations was comparable. Conclusions This study demonstrated that the U100 and U200 URLi formulations are bioequivalent. The accelerated insulin absorption observed for the U100 formulation was maintained with the U200 URLi formulation. Further, the GD were similar for both formulations, supporting the ability of individuals to transfer from U100 to U200 URLi in a 1:1 unit conversion. Trial Registration NCT03616977.

Aims To compare the pharmacokinetic (PK) and glucodynamic (GD) characteristics of ultra rapid lispro (URLi; Eli Lilly and Company, Indianapolis, Indiana), Fiasp® (Novo Nordisk, Bagsvaerd, Denmark), Humalog® (Eli Lilly and Company) and NovoRapid® (Novo Nordisk), in patients with type 1 diabetes (T1D). Materials and Methods This was a randomized, double‐blind, four‐period, crossover study, conducted in 68 patients with T1D. Patients received the same individualized subcutaneous dose of each study drug immediately prior to a liquid test meal. For comparison, 12 healthy subjects received the same test meal. Results URLi had a significantly faster insulin absorption compared to the other insulins tested. Early half‐maximal drug concentration was reached 13 minutes after administration of URLi, which was 6 minutes faster than Fiasp, 13 minutes faster than Humalog, and 14 minutes faster than NovoRapid (all P

Background Ultra rapid lispro (URLi) is a novel insulin lispro formulation developed to more closely match physiological insulin secretion and improve postprandial glucose control. This study compared the pharmacokinetics, glucodynamics, safety, and tolerability of URLi and Humalog® in patients with type 1 diabetes mellitus (T1DM). Methods This was a phase I, two-period, randomised, double-blind, crossover glucose clamp study in younger adult (aged 18–45 years; n = 41) and elderly (aged ≥65 years; n = 39) patients with T1DM. At each dosing visit, patients received either URLi or Humalog (15 units subcutaneously) followed by a 10 h automated euglycaemic clamp procedure. Serum insulin lispro and blood glucose were measured. Results Insulin lispro appeared in serum 6 min faster, and exposure was 7.2-fold greater over the first 15 min postdose with URLi versus Humalog in both age groups. Exposure beyond 3 h postdose was 39–41% lower, and exposure duration was reduced by 72–74 min with URLi versus Humalog in both age groups. Onset of insulin action was 11–12 min faster, and insulin action was 3-fold greater over the first 30 min postdose with URLi versus Humalog in both age groups. Insulin action beyond 4 h postdose was 44–54% lower, and duration of action was reduced by 34–44 min with URLi versus Humalog in both age groups. Overall exposure and total insulin action remained similar for both treatments. URLi and Humalog were well tolerated. Conclusion In patients with T1DM, URLi showed ultra-rapid pharmacokinetics and glucodynamics, with the differences between URLi and Humalog in elderly patients mirroring those in younger adults. ClinicalTrials.gov identifier: NCT03166124. Electronic supplementary material The online version of this article (10.1007/s40262-020-00903-0) contains supplementary material, which is available to authorized users.

Background and objective Ultra rapid lispro (URLi) is a novel insulin lispro formulation developed to more closely match physiological insulin secretion and improve postprandial glucose control. This study compared the insulin lispro pharmacokinetics and glucodynamics, safety and tolerability of URLi and Humalog® after a single subcutaneous dose in patients with type 2 diabetes mellitus (T2DM). Methods This was a phase I, randomised, two-period, two-treatment, double-blind, crossover study in 38 patients with T2DM. At each dosing visit, patients received either 15 units of URLi or Humalog, followed by a 10 h automated euglycaemic clamp procedure. Serum insulin lispro and blood glucose were measured. Results Insulin lispro appeared in the serum 5 min faster (p

URLi is a novel insulin lispro formulation developed to more closely match physiological insulin secretion. This randomized, subject-blind, 3‑period, crossover, 10‑h euglycemic clamp study assessed the pharmacokinetics (PK), glucodynamics (GD) and tolerability after a 7U or 15U dose of URLi or 15U dose of Humalog in 15 healthy Chinese subjects. After a 15U dose, the early 50% tmax occurred 15.3 min (p Disclosure Y. Yu: None. H. Tao: None. J. Leohr: None. E. S. Labell: Employee; Self; Eli Lilly and Company, Stock/Shareholder; Self; Eli Lilly and Company, Johnson & Johnson, Novartis AG, Procter & Gamble Company. D. E. Coutant: Employee; Self; Eli Lilly and Company, Employee; Spouse/Partner; Eli Lilly and Company. H. Liu: None. C. Qian: None.

URLi is a novel insulin lispro formulation developed to more closely match physiological insulin secretion. URLi U200 is a concentrated formulation. This randomized, double-blind, 4-period, replicated crossover, euglycemic clamp study (NCT03616977) assessed pharmacokinetics (PK) and glucodynamics (GD) after 15 U subcutaneous doses of U100 and U200 URLi in 68 healthy subjects (mean ± SD age 39.6 ± 9.6 y). Similar mean PK and GD profiles were observed after dosing U100 and U200 URLi. The formulations were bioequivalent (BE): the 90% CI of ratios of geometric least-square means for the maximum insulin concentration (Cmax) and total exposure (AUC0-∞) were within BE limits of 0.80-1.25. Additionally, the 90% CI for the ratios of geometric means for maximum (Gtot) and total glucose infused (Rmax) were within the BE limits. The time to early half Cmax (early 50% tmax) occurred at approximately the same time for both formulations. Additionally, insulin exposure within the first 15 and 30 minutes was similar for U100 and U200 URLi. The tolerability of the U200 and U100 URLi formulations were comparable. This study demonstrated that U100 and U200 URLi formulations were BE and the faster insulin absorption was maintained in the U200 formulation. The GD were similar and support the ability of patients to transfer from U100 to U200 URLi in a 1:1 unit conversion. Disclosure H. Linnebjerg: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. E.S. LaBell: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company, Johnson & Johnson, Novartis AG. M.A. Dellva: Employee; Self; Eli Lilly and Company. S. Lim: Employee; Self; Eli Lilly and Company. D.E. Coutant: Employee; Self; Eli Lilly and Company. Employee; Spouse/Partner; Eli Lilly and Company. J. Leohr: None. Funding Eli Lilly and Company

URLi is a novel insulin lispro formulation that was developed to more closely match physiological insulin secretion. This 2-site, randomized, 2-period crossover, double-blind study evaluated the pharmacokinetics and glucodynamics during a liquid test meal, after a single 0.2 U/kg SC dose of URLi or Humalog in 13 children, 14 adolescents, 15 adults with T1D. Onset of insulin appearance was faster with URLi vs. Humalog in children (1.1 vs. 6.5 min; p=0.0002), adolescents (1.9 vs. 6.4 min; p=0.001), and adults (0.9 vs. 4.8 min; p=0.004). Early exposure (AUC0-15min) was greater with URLi vs. Humalog: 7-fold (p In summary, URLi accelerated insulin lispro absorption, reduced late exposure and early PPG following a test meal vs. Humalog in children, adolescents, and adults with T1D. Disclosure R. Aronson: None. H. Linnebjerg: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. J. Leohr: None. E.S. LaBell: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company, Johnson & Johnson, Novartis AG. D.E. Coutant: Employee; Self; Eli Lilly and Company. Employee; Spouse/Partner; Eli Lilly and Company. Q. Zhang: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. T. Danne: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Medtronic, Novo Nordisk A/S, Sanofi. R.K. Pollom: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. Funding Eli Lilly and Company

Purpose Ultra rapid lispro (URLi) is a novel insulin lispro formulation developed to more closely match physiological insulin secretion and improve postprandial glucose control. This study compared the pharmacokinetic and glucodynamic parameters of URLi and Lispro (Humalog®) at 3 dose levels in healthy subjects. Methods This randomized, 6-period, subject- and investigator-blind, crossover study included 42 healthy subjects. At each period, subjects received a single subcutaneous dose of 7, 15, or 30 U of URLi or Lispro followed by a 10-h automated euglycemic clamp. Insulin lispro and blood glucose concentrations were measured. Findings Across all 3 doses, insulin lispro appeared in the serum 2–5 min faster, and exposure was 6- to 8-fold greater in the first 15 min, with URLi versus Lispro. Exposure beyond 3 h postdose was 45%–52% lower, and duration of exposure was 67–86 min shorter with URLi versus Lispro for all dose levels. Onset of insulin action was 7–9 min faster and insulin action was ~3-fold greater in the first 30 min with URLi versus Lispro across the dose levels. Insulin action beyond 4 h was reduced by 32%–45%, and duration of action was reduced by 47–67 min, with URLi versus Lispro for all 3 dose levels. Overall exposure and total glucose infused were similar between URLi and Lispro at each dose level. Dose proportionality was observed for maximum and overall exposure after URLi. Less than dose-proportional increases in maximum and total glucose infused were observed and were similar for both URLi and Lispro. Implications URLi exhibited ultra-rapid pharmacokinetic and glucodynamic parameters across all 3 dose levels studied and exhibited dose-proportional increases in exposure in healthy subjects. ClinicalTrials.gov identifier: NCT03286751.

The human inflammatory response can result in the alteration of drug clearance through effects on drug-metabolizing enzymes or drug transporters. In this article, clinical examples are reviewed of how diseases with moderate to severe inflammation can decrease cytochrome P450 (CYP)-mediated drug clearance and alter plasma protein binding. Also examined is how albumin, α-1-acid glycoprotein, drug fraction unbound in plasma, CYP content, and oral clearance can change dynamically with time in response to inflammation.

Safety, tolerability, and pharmacology profiles of LY3127760, an EP4 antagonist, were explored in healthy subjects in a subject/investigator-blind, parallel-group, multiple-ascending dose study. Cohorts consisted of 13 patients randomized to LY3127760, celecoxib (400 mg), or placebo (9:2:2 ratio) for 28 days. LY3127760 was well tolerated; the most commonly observed adverse events were gastrointestinal, similar to celecoxib. LY3127760 increased release of ex vivo tumor necrosis factor alpha after lipopolysaccharide/prostaglandin E2 stimulation when compared with placebo, suggesting a dose-dependent blockade of the EP4 receptor. Compared with placebo, 24-h urinary excretion of prostaglandin E metabolite was modestly increased; prostacyclin metabolite was inhibited; and thromboxane A2 metabolite was unchanged. Effects on sodium and potassium excretion were similar to those of celecoxib. We conclude that LY3127760 demonstrated similar effects on prostacyclin synthesis and renal sodium retention as celecoxib. These data support exploration of LY3127760 at daily doses of 60 mg to 600 mg in phase II trials. This trial's registration number: NCT01968070.

Basaglar®/Abasaglar® (Lilly insulin glargine [LY IGlar]) is a long-acting human insulin analogue drug product granted marketing authorisation as a biosimilar to Lantus® (Sanofi insulin glargine [SA IGlar]) by the European Medicines Agency. We assessed the similarity of LY IGlar to the reference drug product, European Union–sourced SA IGlar (EU-SA IGlar), using nonclinical in vitro and in vivo studies. No biologically relevant differences were observed for receptor binding affinity at either the insulin or insulin-like growth factor-1 (IGF-1) receptors, or in assays of functional or de novo lipogenic activity. The mitogenic potential of LY IGlar and EU-SA IGlar was similar when tested in both insulin- and IGF-1 receptor dominant cell systems. Repeated subcutaneous daily dosing of rats for 4 weeks with 0, 0.3, 1.0, or 2.0 mg/kg LY IGlar and EU-SA IGlar produced mortalities and clinical signs consistent with severe hypoglycaemia. Glucodynamic profiles of LY IGlar and EU-SA IGlar in satellite animals showed comparable dose-related hypoglycaemia. Severe hypoglycaemia was associated with axonal degeneration of the sciatic nerve; the incidence and severity were low and did not differ between LY IGlar and EU-SA IGlar. These results demonstrated no biologically relevant differences in toxicity between LY IGlar and EU-SA IGlar.

URLi is a novel prandial insulin lispro formulation developed to more closely match physiological insulin secretion. This randomized, double-blind, 4-period, crossover study, compared the PK and glucodynamics (GD) of URLi, Humalog® (HL), NovoRapid® (NR) and Fiasp® (FA) after a test meal in 68 adults with T1D, after single individualized SC dose. Twelve healthy subjects provided the “normal” insulin secretory and glucose response to the test meal. URLi had the fastest insulin absorption: early half maximal drug concentration was reached at 13 min (URLi), 19 min (FA), 25 min (HL), and 27 min (NR) (p URLi significantly reduced PPG excursions up to 4h vs. HL and NR (Figure) and URLi’s PPG profile more closely matched that of healthy subjects in the first 2 hours post-meal (Figure). Hypoglycemic events were similar between insulins during the test meal. URLi demonstrated the fastest insulin absorption and the lowest PPG compared to all insulins tested and more closely matched the normal physiological profile of nondiabetic subjects. Disclosure T. Heise: Advisory Panel; Self; Mylan. Research Support; Self; ADOCIA, Boehringer Ingelheim International GmbH, Dance Biopharm Holdings Inc., Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, MedImmune, Mylan, Nordic Bioscience, Novo Nordisk A/S, Pfizer Inc., Poxel, Saniona, Sanofi, Wockhardt, Zealand Pharma A/S. Speaker's Bureau; Self; Eli Lilly and Company, Novo Nordisk A/S. H. Linnebjerg: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. D. Cao: Employee; Self; Eli Lilly and Company. D.E. Coutant: Employee; Self; Eli Lilly and Company. Employee; Spouse/Partner; Eli Lilly and Company. E.S. LaBell: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company, Johnson & Johnson, Novartis AG. S. Reddy: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. E. Zijlstra: Speaker's Bureau; Self; Novo Nordisk A/S. C. Kapitza: Research Support; Self; ADOCIA, Biocon, Boehringer Ingelheim Pharmaceuticals, Inc., Dance Biopharm Holdings Inc., Eli Lilly and Company, Gan & Lee Pharmaceuticals, MedImmune, Mylan, Nestlé, Nordic Bioscience, Novo Nordisk A/S, Poxel SA, Sanofi-Aventis, Wockhardt, Xeris Pharmaceuticals, Inc., Zealand Pharma A/S. J.M. Bue-Valleskey: Employee; Self; Eli Lilly and Company. Q. Zhang: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. J. Leohr: Employee; Spouse/Partner; Accenture. Employee; Self; Eli Lilly and Company. Stock/Shareholder; Spouse/Partner; Accenture. Stock/Shareholder; Self; Eli Lilly and Company. Funding Eli Lilly and Company

URLi is a novel prandial insulin lispro formulation developed to more closely match physiological insulin secretion. This randomized, double-blind, 2 period, crossover, 10 h euglycemic clamp study assessed PK and glucodynamics after one 15U SC dose of URLi and lispro in 41 patients with T1D (mean ± SD age, 32.0 ± 6.8 y; duration of T1D, 17.7 ± 9.3 y; HbA1c, 7.2 ± 0.6%; BMI, 24.8 ± 2.4 kg/m2). After dosing URLi, onset of insulin lispro appearance in the serum was faster (1.3 vs. 6.8 min; p Onset of insulin action was also faster (20 vs. 31 min; p=0.0007) and early action was increased 2.8 fold (p Total insulin lispro exposure and glucose infused during the clamp did not differ and tolerability was similar between URLi vs. lispro. In patients with T1D, URLi accelerated insulin absorption with a reduced late exposure and overall shorter duration vs. lispro, resulting in faster onset of insulin action and reduced late insulin action. Disclosure H. Linnebjerg: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. Q. Zhang: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. E.S. LaBell: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company, Johnson & Johnson, Novartis AG. S. Reddy: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. D.E. Coutant: Employee; Self; Eli Lilly and Company. Employee; Spouse/Partner; Eli Lilly and Company. U. Hoevelmann: None. L. Plum-Moerschel: None. T. Herbrand: None. J. Leohr: Employee; Spouse/Partner; Accenture. Employee; Self; Eli Lilly and Company. Stock/Shareholder; Spouse/Partner; Accenture. Stock/Shareholder; Self; Eli Lilly and Company. Funding Eli Lilly and Company

URLi is a novel prandial insulin lispro formulation developed to more closely match physiological insulin secretion. This randomized, double-blind, 2 period, crossover, 10 hour euglycemic clamp study assessed the pharmacokinetics, glucodynamics and tolerability of insulin lispro after a single 15U SC dose of URLi and lispro (Humalog®) in 38 patients with T2D (mean [±SD] age, 60.0 ± 7.9 years; duration of diabetes, 17.4 ± 7.65 years; HbA1c, 7.4 ± 0.80%; BMI, 30.0 ± 2.97 kg/m2). After dosing URLi, onset of insulin lispro appearance in serum was faster (1.97 vs. 7.28 min; p The onset of insulin action was also faster (31.99 vs. 44.69 min; p No differences were seen in tolerability between URLi and lispro. In patients with T2D, URLi showed an accelerated insulin lispro absorption with a reduced late exposure and overall shorter duration vs. lispro resulting in faster onset of action and reduced late insulin action. This was the first study to investigate URLi in patients with T2D using a euglycemic clamp and results confirmed the faster PK and GD profile of URLi in this population. Disclosure J. Leohr: Employee; Spouse/Partner; Accenture. Employee; Self; Eli Lilly and Company. Stock/Shareholder; Spouse/Partner; Accenture. Stock/Shareholder; Self; Eli Lilly and Company. M.A. Dellva: Employee; Self; Eli Lilly and Company. D.E. Coutant: Employee; Self; Eli Lilly and Company. Employee; Spouse/Partner; Eli Lilly and Company. E.S. LaBell: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company, Johnson & Johnson, Novartis AG. S. Reddy: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. T. Heise: Advisory Panel; Self; Mylan. Research Support; Self; ADOCIA, Boehringer Ingelheim International GmbH, Dance Biopharm Holdings Inc., Eli Lilly and Company, Gan & Lee Pharmaceuticals, Johnson & Johnson, MedImmune, Mylan, Nordic Bioscience, Novo Nordisk A/S, Pfizer Inc., Poxel, Saniona, Sanofi, Wockhardt, Zealand Pharma A/S. Speaker's Bureau; Self; Eli Lilly and Company, Novo Nordisk A/S. G. Andersen: None. E. Zijlstra: Speaker's Bureau; Self; Novo Nordisk A/S. L. Hermanski: None. L. Nosek: None. H. Linnebjerg: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. Funding Eli Lilly and Company

Aims LY2963016 (LY IGlar) and Lantus (IGlar) are insulin glargine products manufactured by distinct processes, but with identical amino acid sequences. This study compared the duration of action of LY IGlar and IGlar in subjects with type 1 diabetes mellitus (T1DM). Materials and methods This was a randomized, double-blind, single-dose, two-period, crossover study. Twenty subjects underwent 42-hour euglycaemic clamps after a single subcutaneous 0.3-U/kg dose of LY IGlar or IGlar. In this study, the duration of action was defined as the time required for blood glucose levels to rise consistently above a predefined cut-off of 8.3 mmol/L (150 mg/dL) from a state of euglycaemia. Blood samples were collected to measure blood glucose for pharmacodynamic (PD) evaluations. Results End of action was reached within 42 hours in 26 of 40 clamps (13 LY IGlar and 13 IGlar). The median duration of action for all subjects was 37.1 and 40.0 hours, and the mean duration of action (calculated using only patients who reached end of action) was 23.8 and 25.5 hours for LY IGlar and IGlar, respectively. The duration of action was demonstrated to be similar between the treatments using time-to-event analysis (log-rank test of equality p = .859). Following administration of LY IGlar and IGlar, the PD parameters of maximum glucose infusion rate (R max) and total glucose infusion during the clamp (G tot) were comparable. Conclusion LY IGlar and IGlar had similar duration of action and comparable PD parameters in subjects with T1DM.

Safety, tolerability, and pharmacology profiles of LY3127760, an EP4 antagonist, were explored in healthy subjects in a subject/investigator‐blind, parallel‐group, multiple‐ascending dose study. Cohorts consisted of 13 patients randomized to LY3127760, celecoxib (400 mg), or placebo (9:2:2 ratio) for 28 days. LY3127760 was well tolerated; the most commonly observed adverse events were gastrointestinal, similar to celecoxib. LY3127760 increased release of ex vivo tumor necrosis factor alpha after lipopolysaccharide/prostaglandin E2 stimulation when compared with placebo, suggesting a dose‐dependent blockade of the EP4 receptor. Compared with placebo, 24‐h urinary excretion of prostaglandin E metabolite was modestly increased; prostacyclin metabolite was inhibited; and thromboxane A2 metabolite was unchanged. Effects on sodium and potassium excretion were similar to those of celecoxib. We conclude that LY3127760 demonstrated similar effects on prostacyclin synthesis and renal sodium retention as celecoxib. These data support exploration of LY3127760 at daily doses of 60 mg to 600 mg in phase II trials. This trial's registration number: NCT01968070.

Basaglar

LY2963016 (LY IGlar) and Lantus (IGlar) are insulin glargine products with identical amino acid sequences. This was a phase 1 single-site, randomized, subject- and investigator-blinded, 4-treatment, 4-period crossover study to compare the pharmacokinetic (PK) and pharmacodynamic (PD) properties of LY IGlar and IGlar at 2 different doses. Fasted healthy subjects were randomly assigned to receive 2 single doses of LY IGlar and IGlar (0.3 and 0.6 U/kg for each product). Blood samples were collected up to 24 hours postdose to assess PK, and a euglycemic clamp lasting up to 24 hours postdose was conducted to assess PD. Twenty-four healthy subjects aged 23 to 52 years participated in the study. The primary PK parameters (area under the concentration versus time curve from 0 to 24 hours [AUC0-24 ] and maximum observed drug concentration [Cmax ]) and PD parameters (total amount of glucose infused during the clamp [Gtot ] and maximum glucose infusion rate [Rmax ]) were not statistically different between LY IGlar and IGlar at either dose. No safety concerns were noted with either drug. The study demonstrated that the PK and PD parameters for LY IGlar and IGlar were comparable following single doses at both 0.3 and 0.6 U/kg.

The Federal Food, Drug, and Cosmetic Act provides FDA and sponsors with a statutory definition of “substantial evidence.” Most often drug effectiveness is established through the conduct of two adequate and well-controlled clinical trials (AWCTs), but there are situations where effectiveness can be sufficiently established through the conduct of a single AWCT. Some of these situations were detailed in FDA’s 1998 guidance document, but there remains some uncertainty for sponsors in determining what cases might merit a single AWCT to meet the substantial evidence standard (depending on indication, drug class, etc). This article is meant as an aid for drug development teams, and regulatory professionals in particular, to navigate successfully through such questions of substantial evidence. We review some of the prerequisites and hurdles for drug approval based on FDA interpretation of substantial evidence standards. Finally, we will provide some suggestions to sponsors, in the context of case examples demonstrating FDA’s interpretation of substantial evidence to support promotional claims.

A data monitoring committee (DMC) is often responsible for monitoring a clinical trial via interim analyses. Those responsible for regulatory strategy and implementation need to be fully engaged with this oversight process to ensure that the trial will achieve its goals with regulators. In the United States, both 21 CFR 312.50 and 21 CFR 312.56 describe sponsor responsibilities related to ongoing clinical trials conducted under an Investigational New Drug Application. Herein, I review the types of trials that may benefit from DMC involvement and provide an example of a DMC clinical trial model. I highlight important features of the roles of the sponsor, DMC, statistician, and the regulatory professional in interim analysis. I review the importance of early planning, knowing when to release interim data, and avoiding type I error rate inflation. Finally, I examine key points that lead to early stopping or study alteration.

Semantic dementia (SD) is a syndrome of progressive loss of semantic knowledge for objects and people. International criteria propose that SD be included in the frontotemporal lobar degeneration syndromes, with progressive non-fluent aphasia and frontotemporal dementia (FTD). However, several related syndromes have been defined that clinically and conceptually share both similarities and differences with SD: fluent progressive aphasia, progressive prosopagnosia, temporal variant of FTD. In order to establish a French consensus for the diagnosis and modalities of evaluation and follow-up of SD, a working group, composed of neurologists, neuropsychologists and speech-therapists, was established by the Groupe de reflexion sur les evaluations cognitives (GRECO). New criteria were elaborated, based on clinical, neuropsychological, and imaging data. They define typical and atypical forms of SD. A diagnosis of typical SD relies on an isolated and progressive loss of semantic knowledge, attested by a deficit of word comprehension and a deficit of objects and/or people identification, with imaging showing temporal atrophy and/or hypometabolism. SD is atypical if the deficit of semantic knowledge is present only within a single modality (verbal versus visual), or if non-semantic deficits (mild and not present at onset) and/or neurological signs, are associated with the semantic loss.

Structure–activity relationship studies are described, which led to the discovery of novel selective estrogen receptor modulators (SERMs) for the potential treatment of uterine fibroids. The SAR studies focused on limiting brain exposure and were guided by computational properties. Compounds with limited impact on the HPO axis were selected using serum estrogen levels as a biomarker for ovarian stimulation.

Studies were designed to evaluate the amaranth method for measuring chlorine dioxide in water. Specifically, the effects of pH and temperature are examined for the amaranth method. The results of interference studies are reported for free available chlorine species, chlorite ion, chlorate ion, iron (III) ion, oxidized manganese, and monochloramine. Additional research focused on selectivity enhancement for chlorine dioxide over free available chlorine using ammonia/ammonium chloride buffer and gas diffusion-flow injection analysis. The results of method detection limit and accuracy and precision studies are reported for measuring chlorine dioxide in the presence of free available chlorine.

A novel immobilization method for the preparation of tetraphenylporphyrin–silica stationary phases is investigated. Stationary phases consisting of immobilized hydroxyphenyl-triphenylporphyrin (HPTPP) on silica are shown to exhibit unmatched selectivity (α=kC70′/kC60′) and improved efficiency for the separation of fullerenes, with α=7 using 100% toluene as the mobile phase. The HPTPP species are immobilized via reaction with glycidoxypropyltrimethoxysilane. The high shape selectivity of columns packed with HPTPP–silicas enables the single step separation of higher molecular mass fullerenes and higher fullerene isomers using strong fullerene solvents as mobile phases.

A crucial step in the isolation of antibiotic substances is establishing whether or not the isolated material represents a new chemical entity. Because of the importance of molecular weight to this process—known as dereplication—mass spectrometry has traditionally played an active role. In this communication a strategy for utilizing liquid chromatography-mass spectrometry (LC/MS) for novelty assessment is described. Crude extracts (20–50 μg) are chromatographed by conventional bore high-performance liquid chromatography (1 mL/min) after which a postcolumn split to divert roughly one-tenth of the sample to the mass spectrometer for molecular weight determination by electrospray ionization (ESI) mass spectrometry. The majority of the effluent is sent to a UV detector and ultimately collected as 1-min fractions for biological testing. As a secondary confirmation of molecular weight, an aliquot of each fraction (< 5%) is taken for analysis by matrix-assisted laser desorption ionization (MALDI). The improved efficiency of this approach over more traditional schemes utilizing off-line fraction collection and conventional ionization methods can be explained by several factors. First, the superior sensitivity of ESI and MALDI means that less material is required for successful analysis. Second, on-line LC/MS optimizes the efficiency of sample transfer and saves both time and labor. Furthermore, the concentration dependence of ESI allows a majority of the material injected for LC/MS to be recovered for biological testing without compromising the signal available for molecular weight determination. As a validation of the above method, crude extracts containing two well-characterized antibiotics—teicoplanin and phenelfamycin—were examined. Results from these analyses are presented along with data from the analysis of a potent unknown antifungal sample.

The preparation, characterization and potential liquid chromatographic applications of various porphyrin-silica stationary phases are reviewed. These new phases are synthesized by covalently linkin...

Packed capillary liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) was used to study the human biotransformation of the anti-emetic drug dolasetron. Urine from subjects given a single 100 mg intravenous dose, containing 14C-labeled dolasetron (50 microCi), was de-salted and concentrated for LC/MS with minimal loss of radioactivity (97% recovery). Aliquots of the de-salted material were injected directly onto a C8 packed capillary column (25 cm x 0.32 mm i.d.) and eluted with an acetonitrile-water gradient, buffered with 1% acetic acid, at a flow rate of 2 microliters min-1. Five metabolites were detected by LC ESI-MS which, yielded molecular mass information but no fragmentation. The identity of each metabolite was confirmed in a subsequent analysis using product ion scans in conjunction with collisionally induced dissociation. Precursor ion scanning was also employed and did not reveal any new biotransformation products. In addition to defining the major routes of biotransformation, the data obtained were compared with a 14C radioprofile prepared in a separate experiment. Qualitative agreement in the two chromatographic profiles enabled the major clusters of radioactivity to be assigned to specific metabolites of dolasetron. An important observation in this comparison was that the signal obtained by ESI did not provide an accurate assessment of the quantity of each metabolite. This was especially true for acidic conjugates (i.e. glucuronides, sulfates), which in the case of dolasetron can exist as zwitterions (no net charge). The results demonstrate the power of packed capillary LC ESI-MS for use in drug biotransformation studies and suggest that caution should be exercised when interpreting relative metabolite abundances from ESI data in the absence of actual reference standards.

Following intravenous administration in the rat, the concentration of MDL 63,246 in plasma was high and long-lasting. Concentrations fell with an apparent three-exponential decay. MDL 63,246 was distributed in a high volume and was cleared quite slowly. The pharmacokinetics of MDL 63,246 in the rat appear to be dose proportional in the dose range of 20 to 50 mg/kg of body weight. When administered subcutaneously, MDL 63,246 was slowly absorbed from the injection site, and the extent of availability was good, being 70.1%. MDL 63,246 was eliminated slowly by both renal and fecal excretion. MDL 63,246 is rapidly and extensively distributed into the tissues. At 0.5 h after drug administration, radioactivity was detected in all organs examined. At 24 h after administration, the total concentrations of radioactivity still increased in some organs which represent a slowly equilibrating compartment, but only the kidneys and liver showed a higher total concentration of radioactivity than plasma. Between 24 and 192 h after treatment, total concentrations of radioactivity decreased very slowly, and finally, apart from brain, all tissues showed higher concentrations than plasma, indicating a very high affinity of MDL 63,246 for tissues. The ratio of the concentration of radioactivity in blood to that in plasma ratio was 0.58, indicating that MDL 63,246 does not diffuse into erythrocytes and that binding to the erythrocyte membrane does not occur. All of these findings appear to correlate with the excellent in vitro and in vivo activities of the compound, suggesting that MDL 63,246 could be therapeutically efficacious at lower dosages and longer treatment intervals than those currently used for vancomycin and teicoplanin.

Both a GC-MS and an LC method have been developed for the simultaneous quantitation of dolasetron and reduced dolasetron in human plasma. The GC-MS method has been utilized in preliminary human pharmacokinetic studies of dolasetron mesylate. Selected ion monitoring was used in these initial studies to obtain the sensitivity and specificity required for quantitation. The GC-MS method has been used in the range of 1-120 ng ml-1 for dolasetron and 1-240 ng ml-1 for reduced dolasetron in plasma. The limit of quantitation for both compounds by GC-MS was 1 ng ml-1. Recently, an LC method has been utilized for quantitation of both compounds on a routine basis. This method utilizes essentially the same sample preparation procedure as the GC-MS method. The LC method has been used in the range of 5-200 ng ml-1 in plasma for dolasetron and reduced dolasetron. In addition, the relationship between the LC and GC-MS methods has been assessed using data obtained from human male volunteers following intravenous administration of 3.0 mg kg-1 of dolasetron mesylate monohydrate.

Of 188 unselected biology students participating in one or both of two tests measuring stereoscopic depth detection ability, 183 (97.3%) were able to see a depth difference at horizontal disparities of 2.3 min arc or smaller. At least 80% could detect depth differences at 30 sec arc disparity. These findings indicate that most people are able to take advantage of the increasing utilization of stereoscopic displays.

The preparation, characterization and potential liquid chromatographic applications of various porphyrin-silica stationary phases are reviewed. These new phases are synthesized by covalently linkin...

The thermodynamics and kinetics of binding of model tripeptides e-N-acetyl-α-N-dansyl-l-Lys-d-Ala-d-Ala (ADLAA) or α-N,e-N-diacetyl-l-Lys-d-Ala-d-Ala (AALAA) to teicoplanin (1a) and a series of sem...

A fragmentation proccess observed for peptides that contain lysine, or other amino acids which possess a free amino group on their sidechain, is reported. The ions generated by this process are found 16 Da below the acylium-type B ions that result from fragmentation at the C-terminal side of lysine or other amine-containing residues in fast-atom bombardment (FAB) mass spectra. These ions, which are referred to as (B—16) ions, permit differentiation between the isobaric amino acids lysine and glutamine in peptide mass spectra. High resolution measurements indicate that (B—16) ions differ in composition from the corresponding B ions by the removal of one oxygen atom. Formation is believed to occur through a cyclization process initiated by nucleophilic attack by the free amino group of the lysine sidechain at the carbon of the acylium ion (B ion). A similar process initiated derectly from the protonated peptide may also occur. Analogous cyclization processes are restricted for glutamine because this residue is comparatively less nucleophilic than lysine (i.e., amide vs amine). Although (B—16) ions have been detected under high energy collisionally induced dissociation, they are formed less readily than by FAB mass spectrometry. A mechanism consistent jwith this observation as well as other experimental evidence is presented to account for the formation of (B—16) ions.

Liquid chromatography—mass spectrometry experiments were carried out to elucidate the structure of the carbamate derivatives formed when polyamines, such as ethylenediamine and putrescine, react with the precolumn fluorophore forming reagent, 9-fluorenylmethyl chloroformate. It has been shown that both primary amine functionalities derivatize with the reagent for polyamines.

Thermospray liquid chromatography—mass spectrometry (TSP LC—MS) was used to determine human urinary metabolites of terfenadine after oral administration of terfenadine tablets. In addition to the two previously identified major metabolites, azacyclonol (MDL 4829) and the ‘acid’ metabolite (MDL 16,455), three additional metabolites were also detected. One of the additional metabolites was identified as the ‘alcohol’ metabolite (MDL 17,523) and the other two were proposed to be an ‘aldehyde’ and a ‘ketone-acid’ metabolites from their TSP mass spectra. The results of this study demonstrated that TSP LC—MS is a useful technique for the study of terfenadine biotransformation.

Three polypeptide neurotoxins (curtatoxins) were isolated from the venom of the spider Hololena curta by reverse-phase high performance liquid chromatography, gel permeation, and ion-exchange chromatography. The purified toxins induced an immediate paralysis in the cricket Acheta domestica that resulted in desiccation and death of the insect within 24-48 h (LD50 congruent to 4-20 micrograms/g); this toxic effect is consistent with irreversible presynaptic neuromuscular blockade. Curtatoxins are a class of sequence-related, cysteine-rich, carboxyl-terminal amidated polypeptides of 36 to 38 amino acid residues. The cysteine residues are conserved at identical sequence positions among these polypeptides and form 4 intramolecular disulfide bonds. Hydropathy calculations show that the toxins have an internal hydrophobic region flanked by hydrophilic and oppositely charged amino- and carboxyl-terminal ends. By analogy to other cysteine-rich arthropod venom proteins, the folded structure of the curtatoxins is likely important for their target specificity and mode of action at the neuromuscular junction.

Semantic dementia (SD) is a syndrome of progressive loss of semantic knowledge for objects and people. International criteria propose that SD be included in the frontotemporal lobar degeneration syndromes, with progressive non-fluent aphasia and frontotemporal dementia (FTD). However, several related syndromes have been defined that clinically and conceptually share both similarities and differences with SD: fluent progressive aphasia, progressive prosopagnosia, temporal variant of FTD. In order to establish a French consensus for the diagnosis and modalities of evaluation and follow-up of SD, a working group, composed of neurologists, neuropsychologists and speech-therapists, was established by the Groupe de réflexion sur les évaluations cognitives (GRECO). New criteria were elaborated, based on clinical, neuropsychological, and imaging data. They define typical and atypical forms of SD. A diagnosis of typical SD relies on an isolated and progressive loss of semantic knowledge, attested by a deficit of word comprehension and a deficit of objects and/or people identification, with imaging showing temporal atrophy and/or hypometabolism. SD is atypical if the deficit of semantic knowledge is present only within a single modality (verbal versus visual), or if non-semantic deficits (mild and not present at onset) and/or neurological signs, are associated with the semantic loss.

The objective of this study was to measure the steady-state cerebrospinal fluid (CSF) concentration of LY450108 and LY451395 (positive modulators of AMPA receptors) in healthy subjects after the administration of 1 mg and 5 mg. Secondary objectives included the evaluation of safety, pharmacokinetics, and steady-state ratio of plasma:CSF concentrations of LY450108 and LY451395 after multiple dosing. This study was an open-label, multiple oral dose study evaluating 1 mg and 5 mg LY450108 and 1 mg and 5 mg LY451395 in 12 (3 subjects per dosing group) healthy subjects, aged 18 to 49 years. Twelve healthy male subjects completed the study. LY450108 and LY451395 were quantifiable in CSF after 1-mg and 5-mg multiple-dose administrations with plasma:CSF ratio of 82:1 and 44:1, respectively. LY450108 and LY451395 1 mg and 5 mg were measured in the CSF. Single and multiple oral doses of LY450108 and LY451395 were determined to be safe and well tolerated in healthy subjects.

A selective estrogen receptor modulator (SERM) is a nonsteroidal compound with tissue specific estrogen receptor (ER) agonist or antagonist activities. In animals, SERMs may produce morphologic changes in hormonally-sensitive tissues like the mammary gland. Mammary glands from female rats given the SERM LY2066948 hydrochloride (LY2066948) for 1 month at ≥ 175 mg/kg had intralobular ducts and alveoli lined by multiple layers of vacuolated, hypertrophied epithelial cells, resembling in part the morphology of the normal male rat mammary gland. We hypothesized that these SERM-mediated changes represented an androgen-dependent virilism of the female rat mammary gland. To test this hypothesis, the androgen receptor antagonist flutamide was co-administered with LY2066948 (175 mg/kg) to female rats for 1 month. Female rats given SERM alone had hyperandrogenemia and the duct and alveolar changes described here. Flutamide cotreatment did not affect serum androgen levels but completely blocked the SERM-mediated mammary gland change. In the mouse, a species that does not have the sex-specific differences in the mammary gland observed in the rat, SERM treatment resulted in hyperandrogenemia but did not alter mammary gland morphology. These studies demonstrate that LY2066948 produces species-specific, androgen-dependent mammary gland virilism in the female rat.

Packed capillary liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) was used to study the human biotransformation of the anti-emetic drug dolasetron. Urine from subjects given a single 100 mg intravenous dose, containing 14C-labeled dolasetron (50 microCi), was de-salted and concentrated for LC/MS with minimal loss of radioactivity (97% recovery). Aliquots of the de-salted material were injected directly onto a C8 packed capillary column (25 cm x 0.32 mm i.d.) and eluted with an acetonitrile-water gradient, buffered with 1% acetic acid, at a flow rate of 2 microliters min-1. Five metabolites were detected by LC ESI-MS which, yielded molecular mass information but no fragmentation. The identity of each metabolite was confirmed in a subsequent analysis using product ion scans in conjunction with collisionally induced dissociation. Precursor ion scanning was also employed and did not reveal any new biotransformation products. In addition to defining the major routes of biotransformation, the data obtained were compared with a 14C radioprofile prepared in a separate experiment. Qualitative agreement in the two chromatographic profiles enabled the major clusters of radioactivity to be assigned to specific metabolites of dolasetron. An important observation in this comparison was that the signal obtained by ESI did not provide an accurate assessment of the quantity of each metabolite. This was especially true for acidic conjugates (i.e. glucuronides, sulfates), which in the case of dolasetron can exist as zwitterions (no net charge). The results demonstrate the power of packed capillary LC ESI-MS for use in drug biotransformation studies and suggest that caution should be exercised when interpreting relative metabolite abundances from ESI data in the absence of actual reference standards.

In recent years, the biotechnological industry has emerged as the major source of new human therapeutic proteins. Although the great majority of these occur naturally as glycoproteins, it has been observed that glycosylation of the recombinantly produced proteins could be fundamental for their in vivo activity (e.g. tissue plasminogen activator, erythropoietin) or, on the contrary, insignificant (e.g. interleukin-1 receptor antagonist, gamma-interferon, granulocyte macrophage-colony-stimulating factor). The inherent heterogeneity of these complex biomolecules presents an exciting challenge in the analytical field for both their structural analysis and the development of suitable analytical methods to guarantee consistency of their production. Owing to this ever increasing therapeutic interest in proteins and glycoproteins, this paper compares the information provided by different analytical techniques (i.e. high-performance liquid chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, capillary electrophoresis, capillary electrophoresis/electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization time-of flight mass spectrometry and high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of flight mass spectrometry when used for the analysis of proteins and glycoproteins. For the sake of simplicity, reference standard proteins and glycoproteins were used as samples.

Rats were each administered a 9 mg/kg iv bolus dose of a 3H-labeled decapeptide anticoagulant, MDL 28,050. Tritium was eliminated rapidly with approximately 50% of the dose recovered in urine within the first 6 hr. Renal excretion accounted for 68% of the dose, whereas fecal excretion accounted for 16% of the dose. Continuous flow fast atom bombardment mass spectrometry was used to identify the major urinary metabolites of MDL 28,050. Trace amounts of parent drug were found, and other biotransformation products indicated that hydrolysis had occurred at four peptide bonds. Two initial sites of hydrolysis were identified as 4I-5P and 6E-7E, which resulted in the peptide fragments Suc-Y-E-P-I-OH + P-E-E-A-Cha-E-OH and Suc-Y-E-P-I-P-E-OH + E-A-Cha-E-OH, respectively. Further metabolism of these fragments resulted in the N-terminal pentapeptide and the C-terminal dipeptide.

This work describes the methodology for the analysis of terfenadine and the acid metabolite of terfenadine in plasma using high-performance liquid chromatography. The use of solid-phase extraction allows the use of robotic or manual sample preparation for the efficient clean-up of terfenadine and terfenadine acid metabolite from plasma. Additional selectivity is obtained through the use of fluorescence detection. For terfenadine, the validated quantitation range of this method is 10.0–84.2 ng/ml with coefficients of variation of 5.7–30%. For terfenadine acid metabolite, the validated quantitation range of this method is 8.2–500 ng/ml with coefficients of variation of 4.1–24%.

The ability to acquire structurally informative daughter ion spectra for individual peptides undergoing separation and analysis by continuous flow fast atom bombardment (CF FAB) is demonstrated. To illustrate the potential of this methodology, tryptic and chymotryptic digests of the 29-residue peptide glucagon were analyzed by CF FAB using mass spectrometric and tandem mass spectrometric detection in consecutive analyses. Daughter ion spectra were recorded using B/E linked scans for the major hydrolysis products observed by liquid chromatography/mass spectrometry. The peptide mixtures were separated by gradient capillary high-performance liquid chromatography with the FAB matrix being added post-column using a coaxial flow interface between the column and flow probe. The entire effluent (3 microl min(-1)) was sampled by the mass spectrometer. Results obtained using less than 300 pmol of digested glucagon indicated several advantages to tandem mass spectrometric detection including the ability to confirm identities for products of enzymatic digestion and the potential use of this method for tandem sequence analysis of peptide mixtures.