Search

The covalent linkage of peptides or protein drugs to human serum albumin (HSA) greatly prolongs their lifetime in vivo but is pharmacologically irrelevant when it irreversibly inactivates them. We retain drug bioactivity by synthesizing a heterobifunctional reagent (MAL-Fmoc-OSu, 9-hydroxymethyl-2-(amino-3-maleimidopropionate)-fluorene-N-hydroxysuccinimide) that generates HSA−Fmoc−insulin on covalent conjugation to the amino group of insulin and the Cys-34 side chain of HSA. HSA−Fmoc−insulin is water-soluble and, upon incubation in aqueous buffers reflecting normal human serum conditions, slowly, spontaneously, and homogeneously hydrolyzes to release unmodified insulin with a t1/2 of 25 ± 2 h. A single subcutaneous or intraperitoneal administration of HSA−Fmoc−insulin to diabetic rodents lowers circulating glucose levels for about 4 times longer than an equipotent dose of Zn²⁺-free insulin. Following subcutaneous administration, onset of the glucose-lowering effect is delayed 0.5−1 h and persists for 12 h. Thus, we present a prototype insulin formulation possessing three desirable parameters:  high aqueous solubility, delayed action following subcutaneous administration, and prolonged therapeutic effect.

Many peptide and protein drugs have a short circulatory half-life in vivo. The covalent attachment of polyethylene glycol (PEG) chains (PEGylation) can overcome this deficiency, but pegylated peptides and proteins are often inactive. In this study, we present a novel PEG−IFNα2 conjugate, PEG40-FMS−IFNα2, capable of regenerating native interferon α2 (IFNα2) at a slow rate under physiological conditions. A 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) containing bifunctional reagent, MAL-FMS-NHS, has been synthesized, enabling the linkage of a 40 kDa PEG-SH to IFNα2 through a slowly hydrolyzable bond. By use of a BIAcore binding assay, the in vitro rate of regeneration of native interferon was estimated to have a half-life of 65 h. Following subcutaneous administration to rats and monitoring circulating antiviral activity, active IFNα2 levels peaked at 50 h, with substantial levels still being detected 200 h after administration. This value contrasts with a half-life of about 1 h measured for unmodified interferon. The concentration of active IFNα2 scaled linearly with the quantity injected. Comparing subcutaneous to intravenous administration of PEG40-FMS−IFNα2, we found that the long circulatory lifetime of IFNα2 was affected both by the slow rate of absorption of the PEGylated protein from the subcutaneous volume and by the slow rate of discharge from the PEG in circulation. A numerical simulation of the results was in good agreement with the results observed in vivo. The pharmacokinetic profile of this novel IFNα2 conjugate combines a prolonged maintenance in vivo with the regeneration of active-native IFNα2, ensuring ready access to peripheral tissues and thus an overall advantage over currently used formulations.

Most low-molecular-weight drugs are short-lived species in the circulatory system, being rapidly eliminated by glomerular filtration in the kidney. However, binding to human serum albumin (HSA) can slow clearance and prolong lifetime profile in vivo. In this study, we have engineered a gentamicin derivative with affinity to albumin by linking three (2-sulfo)-9-fluorenylmethoxycarbonyl (FMS) to three amino groups of gentamicin C1. FMS3-gentamicin associates with HSA with a Ka value of (1.31 ± 0.2) × 10⁵ M^-1. It has less than 1% the antibacterial potency of native gentamicin. Upon incubation at pH 8.5 and 37 °C, the FMS moieties from FMS3-gentamicin undergo slow hydrolysis (t1/2 = 8.0 ± 0.2 h), leading to a linear regeneration of the antibacterial potency with a t1/2 value of 11 ± 0.7 h. FMS3-gentamicin is a long-lived species in the rat circulatory system. Following a single subcutaneous or intravenous administration, it maintains a prolonged pharmacokinetic profile with a peak and a “through” concentration of immuno/antibacterial active gentamicin exceeding 4−5 times the duration obtained by administered native gentamicin. To sum up, an approach aimed at elongating the lifetime of low-molecular-weight drugs in vivo has been examined here with gentamicin. Two to three FMS per mole of compound are to be introduced to obtain an albumin associating affinity of Kd = 7.6−9.2 μM and, hence, to significantly extend the drug's lifetime in situ following administration. By use of this technology, the loss of pharmacological potency with derivatization is of no consequence, since FMS moieties are hydrolyzed and activity is generated at physiological conditions.

In this study we designed, prepared, and analyzed a water-soluble, long-acting insulin derivative whose protracted action in vivo is based on a new principle rather than on slower absorption rates of suspended insulin formulations. To this end, we have prepared (9-fluorenylmethoxycarbonyl-SO3H)3-insulin ((FMS)3-insulin), a derivative having three 9-fluorenylmethoxycarbonyl-SO3H (FMS) moieties covalently linked to the three amino side chains of insulin. (FMS)3-insulin is soluble in aqueous buffers at neutral pH, at a concentration range of 0.15−0.60 mM, and has about 1% of both the biological potency and the receptor-binding affinity of the native hormone. Upon incubation at pH 7.4 and 37 °C, it undergoes a slow hydrolysis with linear regeneration of insulin possessing full biological potency. A single subcutaneous administration of (FMS)3-insulin to streptozocin-treated rats lowered circulating glucose levels for a prolonged period (t1/2 = 30 h). Similarly, intraperitoneal administration of (FMS)3-insulin to healthy rats had a prolonged glucose-lowering effect. In this experimental system, recovery from hypoglycemia proceeded with a t1/2 value of 14 ± 1 h, as compared with t1/2 = 8.0 ± 1 h for native insulin and t1/2 = 10.0 ± 1 h for NPH-insulin. (FMS)3-insulin is more resistant to proteolysis and appears to be nonimmunogenic. On the whole, (FMS)3-insulin represents a prototype version of a water-soluble, long-acting preparation of insulin. It is nearly inactive at the time of administration, and therefore can be administered, at high dose, with no concern for hypoglycemia. Because of its decreased receptor-binding affinity, (FMS)3-insulin evades receptor-mediated endocytosis and degradation and, hence, persists for a long period in the circulation. The insulin constituent of the (FMS)3-insulin conjugate undergoes a slow, spontaneous activation in the circulatory system, manifesting a prolonged glucose-lowering action in vivo. According to the data presented here, (FMS)3-insulin represents a typical prodrug:  a compound which by itself shows only marginal activity but over time it is chemically hydrolyzed to the fully active hormone.

We report that the vanadium ligand L-Glu(gamma)HXM potentiates the capacity of free vanadium ions to activate glucose uptake and glucose metabolism in rat adipocytes in vitro (by 4-5-fold) and to lower blood glucose levels in hyperglycemic rats in vivo (by 5-7-fold). A molar ratio of two L-Glu(gamma)HXM molecules to one vanadium ion was most effective. Unlike other vanadium ligands that potentiate the insulinomimetic actions of vanadium, L-Glu(gamma)HXM partially activated lipogenesis in rat adipocytes in the absence of exogenous vanadium. This effect was not manifested by D-Glu(gamma)HXM. At 10-20 microM L-Glu(gamma)HXM, lipogenesis was activated 9-21%. This effect was approximately 9-fold higher (140 +/- 15% of maximal insulin response) in adipocytes derived from rats that had been treated with vanadium for several days. Titration of vanadium(IV) with L-Glu(gamma)HXM led to a rapid decrease in the absorbance of vanadium(IV) at 765 nm, and (51)V NMR spectroscopy revealed that the chemical shift of vanadium(IV) at -490 ppm disappeared with the appearance of a signal characteristic to vanadium(V) (-530 ppm) upon adding one equivalent of L-Glu(gamma)HXM. In summary, L-Glu(gamma)HXM is highly active in potentiating vanadium-activated glucose metabolism in vitro and in vivo and facilitating glucose metabolism in rat adipocytes in the absence of exogenous vanadium probably through conversion of trace intracellular vanadium into an active insulinomimetic compound. We propose that the active species is either a 1:1 or 2:1 L-Glu(gamma)HXM vanadium complex in which the endogenous vanadium(IV) has been altered to vanadium(V). Finally we demonstrate that L-Glu(gamma)HXM- and L-Glu(gamma)HXM.vanadium-evoked lipogenesis is arrested by wortmannin and that activation of glucose uptake in rat adipocytes is because of enhanced translocation of GLUT4 from low density microsomes to the plasma membrane.

Protein tyrosine kinase (PTK) blockers which competitively inhibit the kinase activity of insulin receptors were synthesized and their properties examined. The best insulin receptor kinase (IRK) inhibitors possess either one hydroxyphenyl ring and two carboxyl groups or two phenyl rings and one carboxyl group. All the inhibitors, except tBoc‐tyrosine aminomalonate, effectively block the IRK‐catalyzed phosphorylation of exogenous substrate, but only partially block receptor autophosphorylation. These PTK blockers inhibit the insulin induced [14C]glucose assimilation into lipids (lipogenesis), but fail to inhibit the anti‐lipolytic effect of the hormone. Only tBocTyr‐aminomalonate was found to inhibit all the effects of insulin measured: insulin‐stimulated phosphorylation of exogenous substrate, IRK autophosphorylation, insulin‐dependent lipogenesis and the insulin‐dependent anti‐lipolytic effect. This inhibitor is the first blocker which is reported to block insulin‐dependent anti‐lipolysis. The inhibitors examined are devoid of general adverse effects since they have no effect on insulin‐independent lipolysis, on [U14C]fructose assimilation or on (‐)isoproterenol‐stimulated lipolysis. These studies suggest that insulin‐dependent lipogenesis and anti‐lipolysis may be mediated by two distinguishable signalling pathways. This study also suggests that PTK inhibitors may become useful tools in the investigation of the signalling pathways of PTKs.

Two methods were used to label insulin receptors covalently with 125I. In the first, an aryl azide derivative of insulin, 125I-labeled 4-azido-2-nitrophenyl-insulin, was synthesized and used to photolabel the binding region of the insulin receptor in rat liver membranes and human placenta membranes. In the second, insulin receptors were purified from rat liver membranes and labeled with 125I by use of chloramine-T; this method presumably has no specificity for the binding region of the receptor. The proteins labeled by both methods were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis after or without reduction by dithiothreitol. The photoaffinity label specifically labeled a single band in both liver and placenta that had an apparent molecular weight of 135,000 after reduction. A band with similar mobility was present in the chloramine-T-labeled preparation, which also contained a second major band with an apparent molecular weight of 45,000. Without reduction, both methods resulted in a single labeled band with an apparent molecular weight of about 310,000. These results indicate that the insulin receptor of both liver and placenta has a subunit of molecular weight 135,000 that binds insulin and that the receptor may be composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds.

Mice immunized to ungulate insulins were found to develop antibodies of two specificities: insulin antibodies that were mostly IgG1 and IgG2 antibodies that acted both as anti-idiotypes to specific mouse insulin antibodies and as antibodies to the insulin receptor. There was a negative association between the presence of anti-idiotypic receptor antibodies and insulin antibodies bearing the specific idiotype; the specific idiotypic antibodies were confined to the early phase of the primary response while the anti-idiotypic receptor antibodies were detected only after the idiotypic antibodies had disappeared. To map the insulin epitope that triggered the specific idiotypic response, we chemically altered the insulin molecule so as to inhibit its interaction with the insulin receptor. The altered insulins triggered high titers of antibodies binding to antigenic determinants on native insulin, but no anti-idiotypic receptor antibodies. Thus, the epitope responsible for the specific idiotypic-anti-idiotypic network was probably the part of the insulin molecule whose conformation is recognized by the insulin receptor.

The cyclic decapeptide, polymyxin B (PMXB), was found to inhibit hypoglycemia in mice receiving exogenous insulin (Amir, S., and Shechter, Y. (1985) Eur. J. Pharmacol. 110, 283-285). In this study, we have extended this observation to rats. Insulin-dependent hypoglycemia in rats is efficiently blocked at a 12:1 molar ratio of PMXB to insulin. This effect is highly specific, as it could not be mimicked by a variety of antibiotics or positively charged substances. Chemical modifications of PMXB have revealed that the ring structure, rather than the tail structure, is important for anti-insulin-like activity. Colistin A, which differs from PMXB by one conservative amino acid substitution in the ring structure, is devoid of this activity. Polymyxin B does not interact with insulin, nor does it alter the rate of insulin absorption and/or degradation, or the ability of insulin to bind to target tissues. This peptide inhibits hypoglycemia by blocking insulin-dependent activation of the hexose transport mechanism, as deduced by in vitro studies. The effect of insulin in stimulating hexose uptake (and subsequent glucose metabolism) in both isolated muscle tissue and adipocytes is blocked with little or no effect on the basal activities of these processes. Colistin A has no significant inhibiting effect. Other insulin-dependent activities, such as inhibition of lipolysis in adipocytes or synthesis of DNA in muscle cells, are not inhibited. It is concluded that PMXB inhibits, in a highly specific manner, the action of insulin in stimulating hexose transport and subsequent glucose metabolism, both in vitro and in the whole animal model.

The effect of oral administration of vanadate, in normalizing blood glucose levels of streptozotocin-treated rats (ST-rats), is further characterized and its mode of action is determined. We have examined the effects of two orally administered doses of sodium metavanadate. High concentrations of orally administered vanadate (0.8 mg/ml in drinking water) reduced blood glucose levels within 2-4 days of application and led to the appearance of hypoglycemia in test animals. Lower concentrations of vanadate (0.2 mg/ml in drinking water) also lowered blood glucose levels within 4 days, but did not lead to hypoglycemia for at least 3 weeks. These effects of vanadate were found to be reversible; hyperglycemia recurred within 2 days after removal of vanadate from the drinking water. In streptozotocin-treated rats receiving low vanadate treatment, circulating levels of vanadate were about 0.8 microgram/ml after 3 weeks of treatment. These rats became anabolic, while rats receiving high vanadate treatment remained catabolic. Subsequent to vanadate treatment, adipocytes derived from ST-rats responded to lower insulin concentrations. In addition, vanadate treatment lowered the increased insulin binding capacity of liver plasma membranes derived from ST-rats. Insulin binding capacity under these conditions approached that of control non-ST-rats. Basal rates of hexose uptake in muscle and liver tissues were doubled in vanadate-treated ST-rats. It is concluded that the oral administration of vanadate leads to normoglycemia by stimulating glucose uptake. Treatment with “low vanadate” leads to the formation of a stable anabolic and normoglycemic state in ST-rats and appears to restore insulin responsiveness of target tissues, without apparent signs of toxicity. Vanadate treatment did not impair either kidney or liver function, as assayed by the measurement of serum urea, creatinine, and glutamic-oxaloacetic transaminase.

The effect of insulin on rat adipocytes was studied in isotonic buffers (pH 7.4) containing NaCl, CaCl2, MgSO4, KCl, and bovine serum albumin but no phosphate or bicarbonate anions. In phosphate- and bicarbonate-free buffers the dose-response curve to insulin is shifted to the right, the effects of the hormone on hexose uptake, glucose metabolism, and inhibition of lipolysis being observed at much higher (nearly 2 orders of magnitude) concentrations of insulin. The insulin binding capacity of the cells is only slightly changed. The dose-response curve for isoproterenol which stimulates lipolysis in the same cell type is almost the same in both Krebs-Ringer bicarbonate buffer and phosphate- and bicarbonate-free buffers. The dose-response curves for agents that mimic the action of insulin such as wheat germ agglutinin or vanadate ions are also shifted to the right. The dose-response curve to insulin can be returned to "normal" by readdition of either bicarbonate or phosphate. Almost complete recovery is obtained at either 10 mM bicarbonate or 24 mM phosphate, respectively. External Ca2+ ions which are not required for the proper action of insulin in fat cells maintained in Krebs-Ringer bicarbonate buffer, become essential for insulin action in bicarbonate-free buffer. The study indicates that depletion of bicarbonate and, to a lesser extent, phosphate anions, interferes with an essential insulin-dependent post-binding event. Also, in bicarbonate-free medium, external Ca2+ ions are essential for insulin-mediated processes. The implications of this study to the mode of action of insulin, and to physiological and clinical states of insulin desensitization are discussed.

In the preceding paper (Shechter, Y., and Ron, A. (1986) J. Biol. Chem. 261, 14945-14950) we have shown that in fat cells, prepared and maintained in an isotonic buffer (pH 7.4) containing neither phosphate nor bicarbonate anions (Buffer A), the dose-response curve to insulin shifted to the right by about 2 logarithms and insulin binding affinity or capacity was only slightly decreased. In the current paper we demonstrate that progressive loss of insulin binding, either by treatment with trypsin or preincubating the cells with isoproterenol, correlates well with the reduced ability of the cells to elicit maximal lipogenesis in response to insulin. We further demonstrate in the "new" system that: the dissociation of labeled insulin from fat cells is not accelerated by the inclusion of unlabeled insulin in the medium; termination of lipogenesis in Buffer A occurs immediately; ligand-induced receptor internalization is grossly defective; and insulin is unable to stimulate lipogenesis at 15 degrees C. The data support the hypothesis that in the new experimental system all measurable binding sites are linked to a coupling mechanism. Each site behaves as an independent, separate entity and there are no site to site interactions. This leads to a linear relationship between binding and bioactivation, lack of negative or positive cooperatively, accelerated rate of termination, defective internalization, a shift to the right in the dose-response curve to insulin, and a lack of insulin response at a lower temperature. In more general terms, the study indicates that all measurable insulin receptors are chemically homogeneous in their potential capability to be coupled to an insulin effector (biologically relevant) system, and they do so under particular experimental conditions.

One of the specific inhibitors of calmodulin action, trifluoperazine, blocked the stimulating action of insulin on 2-deoxyglucose uptake and glucose metabolism. The inhibitory effect of insulin on lipolysis was not altered by the drug. The active (insulin-stimulated) state and the basal state of lipogenesis were inhibited half-maximally at 80 and 550 microM trifluoperazine, respectively. 2-Deoxyglucose uptake was inhibited half-maximally at a trifluoperazine concentration of 70 microM. Other less potent calmodulin inhibitors also inhibited glucose metabolism in fat cells but in a nonspecific manner. The inhibition was noncompetitive and was not altered in Ca2+- free medium. The stimulating activity of wheat germ agglutinin and of sodium vanadate were also inhibited by trifluoperazine. The dose-dependent inhibitions were indistinguishable whether the active (stimulated) state was produced by insulin, wheat germ agglutinin, or vanadate. The data indicate that a late event in the sequence that ultimately leads to enhanced glucose transport activity in fat cells is specifically inhibited by trifluoperazine. The possible involvement of calmodulin or another related Ca2+-dependent regulatory protein in the exocytic (fusion) reaction that recruits glucose-transport activity from storage sites to the plasma membranes is discussed.

Highly fluorescent analogs of insulin and epidermal growth factor were prepared by the covalent attachment of these peptides to alpha-lactalbumin molecules that were highly substituted (i.e., seven to one) with rhodamine molecules. The alpha-lactalbumin was specifically linked to the lysine residue of insulin or to the alpha-amino group of epidermal growth factor. The insulin derivative retained 1.15% of its potency in stimulating glucose oxidation in fat cells but retained about 8.3% of its binding affinity toward receptors. The epidermal growth factor derivative was completely active in binding to fibroblast receptors and 40% as potent as the native hormone in stimulating DNA synthesis. These highly fluorescent derivatives were suitable for the specific visual labeling of receptor sites in viable cells and for measuring the lateral mobilities of the receptor-hormone complexes by fluorescent photobleaching recovery techniques. By these methods it was shown that the hormone-receptor complexes can move laterally in the plane of the plasma membrane with a diffusion coefficient of (3-5) X 10(-10) cm2/sec.

We have prepared a fluorescent conjugate of nerve growth factor (NGF) containing 8--10 rhodamine molecules attached to free carboxyl groups of the protein. This analogue retained full binding capacity toward NGF receptors, full antigenic properties, and the potency to stimulate the differentiation of embryonal chicken sensory ganglia cells in vitro. We have used this analogue to study the mobility and distribution of NGF receptors on embryonal chicken sensory cells from dorsal root ganglia and on a pheochromocytoma cell line (PC-12) that responds to NGF by differentiating along a neuronal pathway. The rhodamine conjugate of nerve growth factor (R-NGF) binds initially to diffusely distributed mobile receptors (D approximately 8 X 10(-10) cm2/sec) on immature sensory and PC-12 cells. At 37 degrees C, the NGF receptor complexes cluster and form immobile visible patches. These patches undergo endocytosis in a process that consumes metabolic energy. Methylamine blocks the formation of visible patches of NGF and the receptors remain dispersed and mobile at 37 degrees C. On differentiated chicken sensory cells, R-NGF binds to diffusely distributed mobile receptors and to aggregated immobile binding sites. These clusters are localized at the tip of the axon, along the axon, and in the main body. The NGF molecules that are internalized at the tip of the axon are transported retrogradely from the peripherey to the cell body.

Incubation of physiological concentrations of 125I-labeled insulin with liver membranes in the presence of anti-insulin IgG results in a 7- to 15-fold increase in the specific binding of the hormone. The low-affinity/high-capacity binding sites are replaced by an apparently homogeneous class of high-affinity sites, and the nonlinear Scatchard plots are converted to linear plots without a change in the maximum number of binding sites. Similarly, the binding of insulin to receptors in 3T3 fibroblasts is increased substantially in the presence of anti-insulin antibody, and the biological activity of subactive concentrations of insulin is enhanced by antibody in these cells. However, the affinity of 125I-labeled epidermal growth factors (EGF) in fibroblasts is not affected by anti-EGF IgG. In adipocytes anti-insulin IgG in the same concentration range only inhibits the binding of insulin and suppresses insulin-mediated glucose oxidation. Monovalent Fab' fragments from anti-insulin IgG inhibit the binding of the hormone, indicating that the enhancement of binding in liver membranes and fibroblasts requires the bivalency of the antibody.