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R E Chance

Publications and source records attributed to R E Chance.

At least 19 recordsLinked to original sources
PubMed2001

Insulin self-association and the relationship to pharmacokinetics and pharmacodynamics.

The treatment of type 1 diabetes requires multiple, daily injections of insulin. While many improvements involving formulation adjustments have been made in an attempt to optimize therapy, clinical experience indicates that the commercially available insulin preparations used for treatment have significant limitations. One principal deficiency relates to poor simulation of the physiological insulin secretion pattern, making achieving normalization of blood glucose concentrations difficult. Endogenous insulin secretion in nondiabetic subjects is characterized by a pulsatile profile that displays multiple, meal-stimulated phases and low basal concentrations between meals and overnight. Optimal diabetes therapy, therefore, requires insulin preparations that display a rapid onset of action with corresponding rapid clearance to provide for meal ingestion as well as preparations that can maintain a sustained, peakless profile for basal requirements. Recent efforts in pharmaceutical research have used the concept of rational-based design of the insulin molecule in an attempt to produce preparations that display more ideal pharmacological profiles. Using detailed structural information obtained from X-ray crystallographic studies to guide design strategies and exploit the nonrestrictive synthetic capabilities of recombinant DNA technology, researchers have prepared a number of insulin analogs that display a reduced propensity towards self-association. Clinical evaluations have shown that these so called "monomeric" analogs better mimic the meal-stimulated pharmacokinetics of insulin secretion observed in nondiabetics. Two monomeric insulin analog preparations have successfully obtained regulatory approval and are now commercially available. Efforts to produce optimized basal-acting insulin analogs have lagged behind. While some of these analogs have been engineered using recombinant DNA technology, design strategies in many cases exploit physicochemical properties of insulin other than self-association. One basal insulin analog has recently received regulatory approval. This paper reviews insulin self-association and its relationship to pharmacokinetics and pharmacodynamics. Particular emphasis is placed on the approaches used to manipulate self-assembly resulting in meal-time insulin analogs that display optimal pharmacological properties. Other design strategies used to develop improved basal insulin preparations are also considered.

Diabetes Mellitus, Type 1↗
PubMed1999

Insulin autoantibodies: more specific than proinsulin autoantibodies for prediction of type 1 diabetes.

Proinsulin autoantibodies (PAA) are a potential alternative to insulin autoantibodies (IAA) for the prediction of type 1 diabetes. We measured the prevalence of PAA and IAA in 179 patients with newly diagnosed type 1 diabetes and 1028 schoolchildren, and compared the potential of these antibodies for disease prediction. Antibodies were measured using a novel microassay in which screening for binding of radio-labelled antigen was followed by competitive displacement. Thresholds for both antigen binding and competitive displacement were selected so that equivalent numbers of patients with type 1 diabetes were identified by the proinsulin and insulin antibody assays. Similar numbers of schoolchildren were found to have insulin and proinsulin binding above the screening threshold but, after competitive displacement, the prevalence of PAA (3.7%) was more than twice that of IAA (1.4%) with 66% sensitivity. Both PAA and IAA were present in 113 (63%) of patients, as against 0.8% of schoolchildren, implying that individuals with both antibodies are at particularly high risk of disease. The majority of samples with IAA or PAA were displaced with both insulin and proinsulin, suggesting that the main epitopes recognized by these antibodies are on the insulin molecule. We conclude that IAA are more specific than PAA for the prediction of type 1 diabetes.

Adolescent↗
PubMed1999

Differences in the cellular processing of AspB10 human insulin compared with human insulin and LysB28ProB29 human insulin.

Cellular metabolism studies were performed comparing human insulin with two rapid-acting analogs, LysB28ProB29 insulin (LysPro) and AspB10 insulin (B10-Asp). B10-Asp bound to isolated hepatocytes at 37 degrees C to a greater extent than LysPro or native insulin, which were equivalent. The rate of degradation was similar for the three materials, resulting in a significant reduction in the degraded/bound ratio for the B10 analog. The processing of membrane-bound material was examined by incubating cells with hormone at 4 degrees C, removing unbound insulin, and incubating the cells at 37 degrees C. Again, binding was greater for B10-Asp versus LysPro or native insulin, with a reduction in the degraded/bound ratio. Hormone internalization and processing was examined by an acid wash of cells incubated with 125I(A14)-labeled hormone to remove surface-bound materials. The processing rate was slower for B10-Asp versus LysPro or native insulin. Cell extraction and examination on molecular-sieve chromatography confirmed that B10-Asp was processed at a slower rate than either LysPro or native insulin. Intact B10-Asp was found in the cell after 4 hours, whereas all native insulin and LysPro were degraded by 90 to 120 minutes. B10-Asp also caused a greater incorporation of thymidine into DNA in cultured cells than LysPro or native insulin, which were similar. These data show that the cellular processing of LysPro is essentially identical to that of native insulin. However, B10-Asp has markedly different properties and is processed much more slowly. The prolonged cell residence time of B10-Asp could contribute to its greater effects on cell growth and mitogenesis.

Animals↗
PubMed1999

Sensitive RIA for the specific determination of insulin lispro.

Insulin lispro is an insulin analog in which the primary sequence has been altered by the inversion of amino acids B28 and B29. To date, it has not been possible to specifically measure insulin lispro in the presence of endogenous insulin because of the high degree of homology between these peptides. However, the specific determination of insulin lispro offers advantages over quantifying total concentrations of immunoreactive insulin. We therefore immunized guinea pigs and screened for antibodies with increased affinity and selectivity for insulin lispro. We prepared a monospecific antiserum by a novel immunoadsorption strategy using despentapeptide insulin. The antiserum was used to develop a competitive RIA for insulin lispro. The RIA has a low limit of quantification (17.2 pmol/L); has no interference from insulin, proinsulin, or C-peptide; and has interassay CVs of 2.6-13.4%. The new RIA is useful for measuring serum concentrations of insulin lispro.

Amino Acid Sequence↗
PubMed1998

Self-association properties of monomeric insulin analogs under formulation conditions.

PURPOSE: The purpose of the current study was to investigate the effects of two important excipients, zinc and m-cresol, on the self-association properties of a series of monomeric insulin analogs. In this way, the effects on formulation behavior of individual amino acid substitutions in the C-terminal region of the insulin B-chain could be compared. METHODS: The self-association of ten insulin analogs was monitored by equilibrium and velocity analytical ultracentrifugation under three different conditions: (i) in neutral buffer alone; (ii) in neutral buffer containing zinc ion; and (iii) in neutral buffer containing both zinc ion and phenolic preservative (a typical condition for insulin formulations). The self-association properties of these analogs were compared to those of human insulin and the rapid-acting insulin analog Lys(B28)Pro(B29)-human insulin. RESULTS: The analogs in the current study exhibited a wide range of association properties when examined in neutral buffer alone or in neutral buffer containing zinc ion. However, all of these analogs had association properties similar to human insulin in the presence of both zinc and m-cresol. Under these formulation conditions each analog had an apparent sedimentation coefficient of s* = 2.9-3.1 S, which corresponds to the insulin hexamer. CONCLUSIONS: Analogs with changes in the B27-B29 region of human insulin form soluble hexamers in the presence of both zinc and m-cresol, and m-cresol binding overrides the otherwise destabilizing effects of these mutations on self assembly.

Amino Acid Sequence↗
PubMed1997

Modifications in the B10 and B26-30 regions of the B chain of human insulin alter affinity for the human IGF-I receptor more than for the insulin receptor.

Inversion of the natural sequence of the B chain of human insulin (HI) from ProB28LysB29 to LysB28ProB29 generates an insulin analogue with reduced tendency to self-associate. Since this substitution increases the homology of insulin to insulin-like growth factor-I (IGF-I), we have examined the affinity of a series of insulin analogues with the general modified structure XaaB28ProB29 HI for binding to both human placental insulin and IGF-I receptors. The XaaB28ProB29 HI series is approximately equipotent to HI in binding to the insulin receptor with the exception of when Xaa = Phe, Trp, Leu, Ile, and Gly (40-60% relative to HI). Substitution with basic residues in the B28 position increased the relative affinity to the IGF-I receptor approximately 1.5-2-fold (ArgB28ProB29 > OrnB28ProB29 = LysB28ProB29). Substitution with acidic residues reduced relative affinity for the IGF-I receptor approximately 2-fold (CyaB28ProB29 = GluB28ProB29 > AspB28ProB29). Combination of AspB10 substitution in conjunction with a modification in the B28-29 position (e.g. AspB10LysB28ProB29 HI) showed an additional 2-fold selective increase in affinity for the IGF-I receptor, suggesting that these two effects are additive. Addition of Arg residues at B31-32, on the backbone of either HI or AspB10 HI, increased affinity for the IGF-I receptor 10 and 28 fold, respectively, compared to HI, confirming the significance of enhanced positive charge at the C-terminal end of the insulin B-chain in increasing selectivity for the IGF-I receptor. This relative increase in IGF-I receptor affinity correlated largely, but not completely, with enhanced growth promoting activity in human mammary epithelial cells. In the case of LysB28ProB29 HI, growth activity correlated with dissociation kinetics from the insulin receptor which were shown to be identical with those of human insulin.

Amino Acid Sequence↗
PubMed1997

Bioavailability and bioeffectiveness of subcutaneous human insulin and two of its analogs--LysB28ProB29-human insulin and AspB10LysB28ProB29-human insulin--assessed in a conscious pig model.

In this study, human insulin was compared with two of its analogs--LysB28ProB29-human insulin and AspB10LysB28ProB29-human insulin-with respect to bioavailability and metabolic effectiveness. Absorption from the subcutaneous site was determined using kinetic parameters from the washout curve, following intravenous infusion of insulin or analog. Absorption was found to be more rapid for the two analogs, with 90% absorption by 100 min for the analogs and by 180 min for insulin. Total absorption was 97 +/- 10% for insulin, 99 +/- 7% for LysB28ProB29-human insulin, and 93 +/- 12% for AspB10LysB28ProB29-human insulin. Bioactivity was assessed from the glucose infusion and using tracer-determined metabolic clearance rates (MCRs) and glucose production rates. The fractional glucose requirements (relative to the total amount infused) increased more rapidly for the two analogs than for insulin, with 50% of the glucose infused by 105 min for both analogs vs. 145 min for insulin. The total amount of glucose required was, however, significantly less (19.7 +/- 1.5 mmol/kg) for AspB10LysB28ProB29-human insulin than for either LysB28ProB29-human insulin (25.9 +/- 3.0 mmol/kg) or human insulin (27.8 +/- 2.6 mmol/kg). The glucose requirements were reflected in a lower MCR for AspB10LysB28ProB29-human insulin but equivalent decreases in the rates of glucose production. Thus both analogs demonstrated more rapid rates of absorption, onset, and termination of action, but were not completely bioequivalent, with AspB10LysB28ProB29-human insulin demonstrating a 25% decrease in bioactivity.

Absorption↗
PubMed1996

Pancreatic polypeptide administration improves abnormal glucose metabolism in patients with chronic pancreatitis.

Chronic pancreatitis (CP) is associated with lowered plasma levels and a blunted nutrient-induced release of pancreatic polypeptide (PP). To investigate the possible role of PP on glucose metabolism, we studied male patients with documented CP (n = 5) and obesity-matched control subjects (NL) (n = 6). Hepatic glucose production (HGP) and overall glucose disposal rates were determined by [3-3H]glucose infusion during a hyperinsulinemic-euglycemic clamp during three separate admissions. Basal rates of HGP were higher in CP patients. In response to an infusion of insulin (60 pmol.m-2.min-1), HGP fell 91 +/- 5% in NL subjects but only 68 +/- 8% in CP subjects (P < 0.05). One month later, the clamp was repeated during the final 2 h of an 8-h infusion of bovine PP (2 pmol.kg-1.min-1). HGP before the insulin infusion and its subsequent suppression (NL: 83 +/- 5%; CP: 86 +/- 15%) were nearly identical between groups. In follow-up studies 1 month after the PP infusion, HGP both basally and in response to insulin alone were similar to the first study. During oral glucose tolerance tests (OGTT) performed 18 h after the PP infusion, subjects with normal (n = 7) baseline OGTT responses showed no effect. All patients with diabetic (n = 3) or nondiagnostic (n = 1) OGTT responses, however, demonstrated lowered mean plasma glucose levels (approximately -2.3 mmol/L; range: -0.6 to -7.2 mmol/L). OGTTs repeated 1 month after the PP treatment showed a return to pretreatment responses. We conclude that chronic pancreatitis accompanied by PP deficiency is associated with partial hepatic resistance both in the basal state and in response to hyperinsulinemia. This impairment is reversed after iv PP administration. PP deficiency may therefore play a role in the development of pancreatogenic diabetes caused by pancreatic injury.

Adult↗
PubMed1996

Characterization of the peptide binding requirements for the cloned human pancreatic polypeptide-preferring receptor.

Traditionally, neuropeptide Y (NPY) receptors have been divided into Y1 and Y2 subtypes based on peptide pharmacology and synaptic localization. Other receptor subtypes have been proposed based on preferences for NPY, peptide YY (PYY), or pancreatic polypeptide (PP). Recently, we discovered a novel human member of this receptor family exhibiting high affinity for PP and PYY. In the current study, we expressed a DNA clone encoding this human PP-preferring receptor [hPP1 (or Y4)] in Chinese hamster ovary cells and performed a peptide structure-activity study. [125I]pPYY bound to homogenates of hPP1-Chinese hamster ovary cells with a Kd of 0.064 +/- 0.006 nM and a Bmax of 244 +/- 12 fmol/mg protein. Human PP inhibited binding with a Ki of 0.023 nM, whereas human PYY (Ki = 0.31 nM) and human NPY Ki = 12 nM) were significantly less potent. Rat, porcine, and bovine PP inhibited binding with similar affinities to human PP, whereas avian PP was substantially less potent (Ki = 1 nM). Deletion of the first four amino acids reduced the affinity of bovine PP to 1 nM. Carboxyl-terminal fragments of NPY and PYY also had reduced potency compared with the native peptides. In addition, deletion of Tyr36-amide produced a substantial reduction in affinity. Pro34-substituted NPY and PYY had modestly increased affinity compared with the native peptides, although Gln34-bPP had similar affinity compared with bovine PP. The carboxyl-terminally derived Y1 antagonist 1229U91 was a very potent (Ki = 0.042 nM) inhibitor of binding to hPP1. Thus, the carboxyl-terminal region of PP seems to be the most important part of the peptide for high affinity binding to hPP1. A few key residues (amino acids 2 and 3) in the amino-terminal region of PP contribute to the high affinity of the native peptide. Thus, features required for peptide recognition by the hPP1 receptor seem to be distinct from the Y1 and Y2 receptor.

Amino Acid Sequence↗
PubMed1995

Structure of a protein in a kinetic trap.

We have determined the structure of a metastable disulphide isomer of human insulin. Although not observed for proinsulin folding or insulin-chain recombination, the isomer retains ordered secondary structure and a compact hydrophobic core. Comparison with native insulin reveals a global rearrangement in the orientation of A- and B-chains. One face of the protein's surface is nevertheless in common between native and non-native structures. This face contains receptor-binding determinants, rationalizing the partial biological activity of the isomer. Structures of native and non-native disulphide isomers also define alternative three-dimensional templates. Threading of insulin-like sequences provide an experimental realization of the inverse protein-folding problem.

Amino Acid Sequence↗
PubMed1994

Improving insulin therapy: achievements and challenges.

Microvascular complications of diabetes can be forestalled by effective glycemic control. However, the inherent limitations of standard subcutaneous insulins reduce their ability to control glycemia without risk of significant hypoglycemia and hyperinsulinemia. Hypoglycemia is unacceptable for most patients and may be dangerous. Hyperinsulinemia is undesirable because it causes weight gain and it has a putative association with atherosclerosis. This paper summarizes the major historical improvements in insulin therapy, and calls attention to the fact that none of the presently available commercial preparations in any combination is capable of simulating the profile of normal insulin secretion--the latter being regarded as the most effective means of normalizing glycemia. For this reason, a variety of new approaches to simulating the pharmacokinetics or glucodynamics of insulin secretion are under investigation. Fast-acting insulin analogues suitable for subcutaneous injection have been developed and appear to mimic the physiological insulin response more closely than standard insulins. Less progress has been made with basal insulins. Intravenous insulin has pharmacodynamic advantages but practical disadvantages of administration. Nasal insulin would be an attractive treatment modality only if its bioavailability could be significantly increased and its safety assured. Other interventions which improve glucose metabolism without necessarily simulating normal insulin secretion are under investigation. These include biosynthetic human C-peptide, insulin-like growth factor-1 and glucagon-like peptide 1 (7-36 amide).

Amino Acid Sequence↗
PubMed1994

Preparation of an insulin with improved pharmacokinetics relative to human insulin through consideration of structural homology with insulin-like growth factor I.

The Diabetes Control and Complications Trial has emphasized the need for improved control of blood glucose as a means to diminish long-term complications of diabetes. LysPro-insulin is an analog of human insulin whose design was modeled on structural homology with insulin-like growth factor I. An analysis of the structural conformation of insulin suggested that an inversion of amino acids B28 and B29 in the C-terminus of the B chain could yield an insulin analog with a faster onset of biological action. This insulin analog has proved to be virtually identical to human insulin in action, with one important exception. LysPro-insulin has demonstrated an improved time course of action in control of a mealtime glucose elevation. This offers the opportunity for improved convenience and safety for patients with insulin-dependent diabetes mellitus.

Amino Acid Sequence↗
PubMed1993

Research, development, production, and safety of biosynthetic human insulin.

This paper provides some historical aspects on the research and development of Humulin (rDNA origin), the first human health-care product derived from rDNA technology more than a decade ago. Also referred to as biosynthetic human insulin, Humulin is currently produced via the human proinsulin route, using an Escherichia coli fermentation process. The authenticity, high purity, and safety of BHI has been investigated and verified by a complex battery of analytical and physicochemical methods. The daily treatment of more than two million diabetic patients worldwide with this rDNA human insulin not only demonstrates the value of rDNA technology in providing an important medical product, it is assurance that diabetic patients will have unlimited supplies of this vital hormone as well as potential analogue refinements.

Amino Acid Sequence↗
PubMed1992

Detection of an equilibrium intermediate in the folding of a monomeric insulin analog.

To determine the conformational properties of the C-terminal region of the insulin B-chain relative to the helical core of the molecule, we have investigated the fluorescence properties of an insulin analog in which amino acids B28 and B29 have been substituted with a tryptophan and proline residue respectively, ([WB28,PB29]insulin). The biological properties and far-UV circular dichroism (CD) spectrum of the molecule indicate that the conformation is similar to that of native human insulin. Guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation of the analog as monitored by CD intensity at 224 nm indicates a single cooperative transition with a midpoint of 4.9 M GdnHCl. In contrast, when the equilibrium denaturation is observed by steady-state fluorescence emission intensity at 350 nm, two distinct transitions are observed. The first transition accounts for 60% of the observed signal and has a midpoint of 1.5 M GdnHCl. The second transition roughly parallels that observed by CD measurements with an approximate midpoint of 4.5 M GdnHCl. The near-UV CD spectrum, size-exclusion, and ultracentrifugation properties of [WB28,PB29]insulin indicate that this analog does not self-associate in a concentration-dependent manner as does human insulin. Thus, the observed fluorescence changes must be due to specific conformational transitions which occur upon unfolding of the insulin monomer with the product of the first transition representing a stable folding intermediate of this molecule.

Amino Acid Sequence↗
PubMed1992

(A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin.

The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide. Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin. A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain. To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free. Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein. By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity. Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding. Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin. The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin. Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation. The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin.

Amino Acid Sequence↗
PubMed1992

Improved insulin stability through amino acid substitution.

Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.

Amino Acid Sequence↗