In vivo demonstration of insulin-receptor defect with 123I-labeled insulin and scintigraphic scanning in severe insulin resistance.

OBJECTIVE: Insulin-receptor function in humans is usually studied in vitro on readily available cells, e.g., erythrocytes and fibroblasts. Although these cells are not metabolically important targets for insulin action, information derived from them are often taken as representative of other tissues. The aim of this study was to investigate insulin receptors in vitro on erythrocytes and in vivo on one of the main insulin-target organs, the liver. RESEARCH DESIGN AND METHODS: A 16-yr-old girl affected by severe insulin resistance was identified. Insulin receptor binding was measured on the erythrocytes of the patient and of 6 nondiabetic volunteers. The biodistribution of 123I-labeled insulin was studied in vivo by scintigraphic scanning in the insulin-resistant patient and in 10 nondiabetic volunteers. RESULTS: Erythrocytes of this patient displayed a markedly reduced [125I]insulin binding. In vivo 123I-insulin biodistribution was characterized by lack of hormone uptake by the liver (4 vs. 21% of the injected dose in control subjects) contrasting with intense accumulation of radioactivity in the kidneys. CONCLUSIONS: Our studies show that defects of insulin binding can be directly demonstrated in vivo on liver receptors with a noninvasive technique with low radiotoxicity.

Imaging of the buffering effect of insulin antibodies in the autoimmune hypoglycemic syndrome.

Insulin autoimmune hypoglycemia is characterized by recurrent hypoglycemia and high levels of immunoreactive insulin in the presence of insulin autoantibodies. The mechanisms inducing hypoglycemia are largely unknown. An [123I]insulin scintigraphic scanning was performed to directly demonstrate the effect of antibodies on insulin biodistribution in one patient with this syndrome both before and after treatment. The patient had insulin autoantibodies IgG3 lambda, which had a single site dissociation constant (Kd = 10(-7) mol/L, by Scatchard analysis), a very fast dissociation rate of immune complexes, and a very rapid association of [125I]insulin. Insulin receptors on red blood cells were down-regulated. The [123I]insulin scintigraphic study imaged the buffering effect of antibodies on insulin bioavailability. [123I]Insulin was not removed from the blood, and no liver or kidney uptake of the hormone occurred. The frequency and severity of hypoglycemic episodes required treatment. Insulin antibody levels decreased and [123I]insulin biodistribution improved after treatment with plasmapheresis and prednisone. Improved hormone bioavailability was further evidenced by the reduction in the hypoglycemic delay after i.v. insulin from 90 min before any treatment to 60 min after plasmapheresis and 30 min after steroid administration. Glucose tolerance was normal after treatment. Plasmapheresis followed by steroid treatment can lower the insulin antibody concentration, abolish severe hypoglycemia, and improve insulin biodistribution and glucose tolerance in insulin autoimmune hypoglycemia.

Enhancement of ricin toxin A chain immunotoxin activity: synthesis, ionophoretic ability, and in vitro activity of monensin derivatives.

Site-selective toxin delivery was achieved by coupling monoclonal antibody to the A chain subunit of ricin (RTA-IT). The cell-killing potency of RTA-IT can be drastically increased in vitro by using ionophores such as monensin. To reduce the intrinsic toxicity of monensin and to enhance its in vitro and in vivo activity, we synthesized 7 derivatives characterized by different lipophilicities. These derivatives were also analyzed for ionophoretic activity on intact cells, toxicity, and RTA-IT-enhancing activity. Two different RTA-IT were assayed on a human leukemia cell line. A correlation between lipophilicity, ionophoretic activity, and RTA-IT enhancement was observed. The compounds with the highest polar charge showed low intrinsic toxicity, revealed moderate ionophoretic activity, and were able to enhance RTA-IT only at high concentrations, whereas more lipophilic compounds (with a C28 tail or a phenyl group) showed significant ionophoretic activity and good enhancing properties.

Poly(ethylene glycol)-human serum albumin-paclitaxel conjugates: preparation, characterization and pharmacokinetics.

Paclitaxel has been found to be very effective against several human cancers, such as ovarian, breast and non-small cell lung cancer and has received marketing approval for metastatic cancers. One of main problems with its use is its poor solubility, which makes irritant solubilitazion agents necessary. In previous research we demonstrated that linkage to human serum albumin (HSA) was useful to increase the in vivo performance of paclitaxel. In this article, in order to improve stability and solubility of paclitaxel conjugate, we linked covalently a monomethoxy poly(ethylene glycol) (mPEG) chain to HSA. New thioimidate mPEG derivatives, highly reactive and stable, were used and two different conjugates (with PEG of molecular mass 2 or 5 kDa) were prepared, purified and characterized. The antitumor activity of the free drug and conjugates was tested on three different tumor cell lines. The PEG grafted conjugates maintained high cytotoxicity, similar to that of ungrafted conjugates, with efficient cell binding and internalization followed by release of the drug inside the cell. The changes in pharmacokinetics and distribution of radio-labelled conjugates were evaluated by i.v. administration to mice and compared with those of the free drug and ungrafted conjugates. The total clearance was reduced (from 3.6 ml/h for free drug to 2.9, 1.97 and 1.41 for ungrafted, 2 and 5 kDa PEG conjugates, respectively). Organ uptake was reduced, in particular by liver and spleen.

Preparation, characterization and properties of sterically stabilized paclitaxel-containing liposomes.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, approved by the FDA for the treatment of ovarian and breast cancers. Due to its low solubility in water, it is clinically administered dissolved in Cremophor EL, (polyethoxylated castor oil) and ethanol, which cause serious side effects. Inclusion of paclitaxel in liposomal formulations has proved to be a good approach to eliminating this vehicle and improving the drug's antitumor efficacy. We prepared different conventional and PEGylated liposomes containing paclitaxel and determined encapsulation efficiency, physical stability and drug leakage in human plasma. The best conventional liposome formulation was composed of ePC/PG 9:1, while for PEGylated liposomes the best composition was ePC/PG/CHOL/PEG(5000)-DPPE 9:1:2:0.7. PEGylated liposomes were found to be less stable during storage than the corresponding conventional liposomes and to have lower drug release in human plasma at 37 degrees C. In vitro cytotoxic activities were evaluated on HT-29 human colon adenocarcinoma and MeWo melanoma cell lines. After 2 and 48 h, conventional liposomes had the same cytotoxicity as free paclitaxel, while PEGylated liposomes were as active as free drug, only after 48 h. Pharmacokinetics and biodistribution were evaluated in Balb/c mice after i.v. injection of paclitaxel, formulated in Cremophor EL or in conventional or in PEGylated liposomes. Encapsulation of paclitaxel in conventional liposomes produced marked differences over the free drug pharmacokinetics. PEGylated liposomes were long-circulating liposomes, with an increased t(1/2) beta 48.6 h, against t(1/2) beta 9.27 h of conventional liposomes. Biodistribution studies showed a considerable decrease in drug uptake in MPS-containing organs (liver and spleen) at 0.5 and 3 h after injection with PEGylated compared to conventional liposomes.

Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing water-soluble prodrugs of paclitaxel.

Paclitaxel (Taxol) is a diterpenoid isolated from Taxus brevifolia, used clinically for the treatment of ovarian and breast cancer. Due to its aqueous insolubility it is administered dissolved in ethanol and Cremophor EL (polyethoxylated castor oil), which has serious side effects. In order to eliminate this vehicle, in previous work we entrapped paclitaxel in conventional and in polyethylene glycol coated liposomes. However, in neither formulation did we obtain satisfactory entrapment efficiency. In this study we increased the paclitaxel concentration entrapped in liposomes by incorporating different water-soluble prodrugs, such as the 2'-succinyl, 2'-methylpyridinium acetate and 2'-mPEG ester paclitaxel derivatives, in the lipid vesicles. Liposomes containing 2'-mPEG (5000)-paclitaxel showed the best performance in terms of stability, entrapment efficiency and drug concentration (6.5 mgml(-1)). The in vitro cytotoxic activity of this liposomal prodrug was similar to that of the parent drug. The pharmacokinetic parameters for the free and for the liposomal prodrugs fitted a bi-exponential plasma disposition. The most important change in pharmacokinetic values of the prodrug vs. the free drug liposomal formulations was t(1/2)beta, plasma lifetime, which was longer in liposomes containing 2'-mPEG (5000)-paclitaxel.

99mTc-2GAM: a tracer for renal imaging.

We propose a renal imaging agent, the 99mTc complex of the bidentate-N,S chelate N-(mercaptoacetyl)glycine (99mTc-2GAM), with the imaging characteristics of 99mTc-DMSA but a faster kidney uptake; chemical evidence supports the formulation of 99mTc-2GAM as [Tc(v)(O)(GAM)2]-. After biodistribution and toxicity studies in animals, 99mTc-2GAM was evaluated in five normal volunteers. 99mTc-2GAM is rapidly cleared from the blood (t1/2 = 9 min) and 50% of the ID is excreted in the urine in the first 2 h. Dynamic data show a rapid renal uptake that increases up to 1 h with no significant wash-out between 1 and 8 h. The uptake in each kidney ranges from 11.3% to 20.7% ID. Low, stable liver uptake is observed. No significant activity is detected in other organs. We showed no differences between 99mTc-2GAM and 99mTc-DMSA compared in three patients with unilateral kidney disease. We conclude that 99mTc-2GAM has good practical and dosimetric features for renal imaging.

Single-step purification of immunotoxins containing a high ionic charge ribosome inactivating protein clavin by carboxymethyl high-performance membrane chromatography.

High-performance membrane chromatography (HPMC) and HPLC hydroxyapatite chromatography were compared for their efficiency in purifying immunotoxins (ITs) containing the ribosome-inactivating protein clavin, which is characterized by a high anionic charge and a low molecular mass. Both methods efficiently removed unreacted clavin from the conjugate crude mixture, but only the cation-exchange HPMC allowed efficient single-step separation of the unreacted monoclonal antibody (mAb) from ITs obtained by different coupling procedures.

Melphalan monitoring during hyperthermic perfusion of isolated limb for melanoma: pharmacokinetic study and 99mTc-albumin microcolloid technique.

BACKGROUND: The kinetics of melphalan leakage from extracorporeal fluid to the peripheral blood was studied in ten patients undergoing hyperthermic isolation perfusion of the lower limbs as an adjuvant treatment in high-risk melanoma. MATERIALS AND METHODS: Systemic leakage was monitored by a new technique using 99mTc-albumin microcolloid. Serial samples were drawn from a peripheral vein and from the perfusion circuit during surgical treatment and analysed by HPLC. RESULTS: The leakage measured with 99mTc-albumin microcolloid ranged from 1.5 to 18%/h (mean 8%/h). The average concentrations in the perfusate were 200-300-fold those found in the systemic circulation. A good correlation (R=0.945) was obtained between systemic AUC (0 to 1 hour) and leakage measured through the 99mTc procedure. Negligible toxicity was found and the survival rate yielded 92% of objective response. CONCLUSION: By studying the pharmacokinetic data of melphalan in the circuit and in the systemic circulation, we were able to validate the 99mTc procedure used during clinical perfusion. Moreover, considering the efficiency of the system as well as the minimum toxicity and the high survival rate, a reduction of perfusion time may be considered.

Preparation and biological characterization of conjugates consisting of ricin and a tumor-specific non-internalizing MAb.

MOv18, a non-internalizing monoclonal antibody (MAb) with restricted tumor specificity, was conjugated to ricin toxin (RT). According to their ability to bind to galactose residues of Sepharose 6B, the immunoconjugates were fractionated into Bound and Unbound MOv18-RT. The two conjugates could be distinguished by SDS-PAGE, in vivo toxicity and agglutination capability. When the binding activity of both fractions was compared by solid-phase RIA to that of native MAb, it proved to be similar on the relevant target cells but significantly increased on the non relevant cells. On the latter, galactose totally cancelled the binding of the Unbound immunoconjugate, whereas it could only partially reverse that of the Bound MOv18-RT. By in vitro cytotoxic activity, either in the presence or absence of galactose, only a slight selectivity for relevant versus non-relevant target cells was observed for both conjugates. It seems that in the presence of a MAb which is incapable of internalization, the conjugate cytotoxicity could only be attributed to RT, with a loss of the MAb's specificity.

Toxin-targeted design for anticancer therapy. I: Synthesis and biological evaluation of new thioimidate heterobifunctional reagents.

In an effort to obtain a more potent and specific immunotoxin for cancer therapy, we designed a series of heterobifunctional linkers characterized by a thioimidate group linked to a S-acetyl thiol (4, 5) or substituted aryldithio group (6-10). These ligands were synthesized by a Pinner-type process from the corresponding nitrile derivatives obtained by thiol-disulphide exchange reaction, reaction with substituted benzene-sulphenyl chloride, or other known procedures. To check the reagent of choice for immunoconjugate preparation, we studied thioldisulphide exchange kinetics between the intermediate nitrile derivatives and cysteine. Among the tested aryldithio derivatives (6-10), we selected ethyl 3-(4-carboxamido-phenyldithio)propionthioimidate (CDPT, 9) for further studies. By analyzing the rate of incorporation of the linkers 4, 5, and 9 in a model immunoglobulin G protein, we found similar results with CDPT 9 and ethyl S-acetyl 3-mercaptopropionthioimidate ester hydrochloride (AMPT, 5) because both reagents showed a linear correlation between the number of introduced thiol groups and factors such as time and protein and reagent concentrations. Comparison of the two acetylthio-derivative ligands 4 and 5 showed that AMPT 5 was more stable toward deacetylation than ethyl S-acetyl 2-mercaptopropionthioimidate ester hydrochloride (AMAT, 4). By comparing the kinetic and biological parameters of seven new thioimidate linkers, we found that two of these (CDPT and AMPT) could be superior ligands for protein-protein conjugation. They offer advantages over the commercially available compounds, such as minimal perturbation of the protein structure, controlled reactivity, and good stability.

Toxin-targeted design for anticancer therapy. II: Preparation and biological comparison of different chemically linked gelonin-antibody conjugates.

To obtain more potent immunotoxins for anticancer therapy a gelonin-AR3 antibody immunoconjugate was prepared with different new linkers and coupling procedures. The gelonin was derivatized with the heterobifunctional thioimidate linkers ethyl-acetyl-3-mercaptopropionthioimidate (AMPT) and 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT), and with the succinimidyl type reagents N-succinimidyl-3-(4-carboxamidophenyldithio)propionate (SCDP) and N-succinimidyl-S-acetyl thiolacetate (SATA). The biological activity of gelonin modified with different linkers (AMPT, CDPT, SCDP, SATA) was determined by a rabbit reticulocyte assay. We found that AMPT was the molecule of choice to derivatize the toxin, confirming the preferability of thioimidate linkers. The monoclonal antibody Mab was derivatized with CDPT and SCDP. Then the following immunoconjugates were prepared with different procedures: Mab-CDPT with gelonin-AMPT; Mab-CDPT with gelonin-CDPT; Mab-SCDP with gelonin-SATA. To verify whether selection of the most suitable coupling procedure could affect the antitumoral activity of the gelonin-AR3 immunoconjugate, the three immunotoxins were tested on target HT-29 human colon carcinoma cells versus nontarget MeWo cells. The gelonin immunoconjugate linked via the AMPT-CDPT thioimidate reagents showed highest antitumoral activity as well as best selectivity for the target cells.

Antitumoral activity of liposomes and immunoliposomes containing 5-fluorouridine prodrugs.

Liposomes and immunoliposomes containing cytotoxic agents may be highly efficacious in intracavity therapy of malignancies confined principally to the peritoneal cavity. To assess the feasibility of this locoregional treatment, we prepared two derivatives of 5-fluorouridine (5-FUR), a highly cytotoxic metabolite of 5-fluorouracile, and incorporated them into REV liposomes, prepared with the reverse phase evaporation method. Encapsulation efficiency, drug leakage, and stability were determined, and size analysis and differential scanning calorimetry were carried out to evaluate the drug delivery potential of liposomes containing 5'-palmitoyl-5-FUR, 5'-succinyl-5-FUR, or the parent drug 5-FUR. The most suitable drug for encapsulation, in terms of minimum leakage and encapsulation efficiency, was 5'-palmitoyl-5-FUR, which differential scanning calorimetry indicated as being firmly anchored to the lipid bilayer. Thus, 5'-palmitoyl-5-FUR was chosen to prepare a chemotherapeutic liposome-monoclonal antibody conjugate (immunoliposome). The covalent linkage between antibody and liposome was realized by coupling the thiolated monoclonal antibody AR-3 with REV liposomes, containing N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine. The cytotoxic activity of drug-bearing liposomes and immunoliposomes was evaluated on the HT-29 human colon adenocarcinoma cell line; the immunoliposomes had higher cytotoxicity than liposomes or 5-FUR. To explore the potential of these drug formulations in anticancer therapy, we ip injected liposomes or immunoliposomes into athymic mice ip grafted with human HT-29 cell line. In this mouse model, the immunoliposome containing 5'-palmitoyl-5-FUR displayed the best antitumoral activity, since on day 27 postgraft only 5% of residual tumor mass was present, compared to control mice; there was a close relationship between exposure time of tumor tissue to the drug and antitumor potency.

Pharmacokinetics of an antibody-ricin conjugate administered intraperitoneally to mice.

Immunotoxins have been extensively studied for the treatment of neoplasias; their intracavitary administration could be useful for the therapy of tumors confined to the pleural or peritoneum spaces. To study the feasibility of this "locoregional" treatment, a pharmacokinetic study of immunotoxins delivery is necessary. Ricin, a plant toxin extracted from the seeds of Ricinus communis, has often been used in immunoconjugates for its high activity; nevertheless, appropriate strategies have been necessary to limit the aspecific toxicity. We previously prepared a AR-3-ricin immunotoxin lacking the ability to bind galactosidic cell surface residues, a so-called sterically blocked immunotoxin. The monoclonal antibody AR-3, an IgG1 specific to the CAR-3 antigen, was able to recognize human colorectal adenocarcinomas. Preclinical trials in nude mice, intraperitoneally grafted with the target neoplasia, showed that this immunotoxin suppressed tumor growth without showing any undesirable ricin toxicity. In the present work we report the pharmacokinetic properties of this immunotoxin, showing the in vivo stability and a relatively long blood survival. With a biodistribution study in tumor-bearing mice, we demonstrate that in tumor-invaded tissues, the concentration of the specific AR-3-ricin immunotoxin was higher and progressively increased in a multiple-dose regimen. In contrast, an irrelevant immunotoxin behaved differently because it did not show specific tumor uptake. Moreover the pharmacokinetic data reported in this work improve the potential for "locoregional" treatment of malignancy with blocked immunotoxins.

A new approach in the synthesis of immunotoxins: ribosome inactivating protein noncovalently bound to monoclonal antibody.

This study describes the synthesis of a new generation of immunotoxins made by a noncovalent interaction between a monoclonal antibody derivatized with a dichlorotriazinic dye and the ribosomal inhibitor protein gelonin. The scheme of preparation has several advantages with respect to the traditional methods, which used heterobifunctional cross-linkers, such as a higher overall yield of production and the homogeneity of the obtained conjugate. Moreover, because no chemical derivatization of the gelonin was required, the unconjugated ribosome inactivating protein was recovered unaltered and therefore can be reused in other synthetic processes. This immunoconjugate was stable when tested in mouse serum and showed an interesting slow elimination rate when administered intravenously in mice. Although a high dye derivatization degree induced a modification of the specificity of the monoclonal antibody, the native specificity was restored after conjugation with gelonin. Furthermore the noncovalent linkage did not affect the gelonin inhibitory activity; in fact, the specific cytotoxic activity seemed to be similar to that of other disulfide-linked immunotoxins previously prepared in our laboratories.

Insulin receptor antibodies inhibit insulin uptake by the liver: in vivo 123I-insulin scintigraphic scanning and in vitro characterization in autoimmune hypoglycemia.

BACKGROUND: Insulin receptor antibodies can induce severe hypoglycemia or insulin resistance in rare autoimmune syndromes. In vitro properties of these antibodies occasionally explain the clinical features of the syndrome, but direct evidence of their in vivo activity is poor. We studied a 58-year-old male with rheumatoid arthritis who presented with hypoglycemic coma. METHODS AND RESULTS: Antibodies were detected by inhibition of 125I-insulin binding to human insulin receptor-3T3 cells by the patient's serum. By immunofluorescence, they were immunoglobulin G of all four subclasses, immunoprecipitated insulin receptors from biotin-labeled cells, and triggered phosphorylation of the beta subunit of the insulin receptor. Insulin binding on the patient's red blood cells was markedly reduced. A biodistribution study after intravenous 123I-Tyr A14 insulin showed a marked inhibition of tracer uptake by the liver, reaching 10% of the injected dose (controls, mean +/- SD, 21.1 +/- 1.7%; n = 10). Time activity curves generated on the liver and on the heart were parallel, with a T1/2 of 11.5 minutes for both, suggesting that no specific uptake occurred in the liver, where tracer activity represented only the blood pool. Clearance of insulin from the blood was indeed slower than in controls and mainly occurred through the kidneys. Analysis of plasma 123I-insulin immunoreactivity and trichloroacetic acid precipitate showed that insulin degradation did not occur as in normal controls. CONCLUSIONS: In this patient with hypoglycemic syndrome, insulin receptor antibodies with in vitro insulin-like activity are capable of blocking in vivo the access of insulin to the liver receptor compartment, as directly demonstrated by the markedly altered biodistribution of intravenously injected 123I-insulin.

From conventional to stealth liposomes: a new Frontier in cancer chemotherapy.

In this review, the different types of liposome used in medicine, in particular in the field of antitumor therapy, are focalised, emphasizing their structures, pharmacological action, pharmacokinetics and biodistribution, toxicity profiles and in the main clinical applications. The first-generation liposomes (conventional liposomes) comprised a liposome-containing amphotericin B, Ambisome, and Myocet, doxorubicin-containing liposome used in clinical trials to treat metastatic breast cancer. The last generation liposomes were pegylated liposomal doxorubicin (Caelix), called "stealth liposomes" because of their ability to evade interception by the immune system, characterized by very long-circulation half-life, favourable pharmacokinetic behaviour and specific accumulation in tumor tissues.

Reverse perfusion pattern of Tc-99m MIBI heralding the development of myocardial infarction.

In a patient with sporadic atypical chest pain associated with dyspnea, stress Tc-99m MIBI imaging showed normal perfusion and inferoposterior hypoperfusion on the resting study. Although this reverse perfusion pattern was considered artifactual, the patient later had an acute myocardial infarction involving the same areas. Postinfarction stress Tc-99m MIBI imaging showed a nonreversible defect in the same area that, in the earlier study, showed a reverse perfusion pattern. The authors hypothesize that partial stenosis of the related artery with some nontransmural myocardial necrosis at the time of the initial study may be a possible cause of this peculiar Tc-99m MIBI perfusion pattern.

A new 'solid phase' procedure to synthesize immunotoxins (antibody-ribosome inactivating protein conjugates).

A method to produce immunotoxins (conjugates comprising of a monoclonal antibody and toxin) using ribosome inactivating protein anchored on an affinity gel derivatized with triazinic dye is described. The adsorbed toxins were activated with 2-imino-thiolane and then conjugated to monoclonal antibody activated by SPDP. The "heterogeneous phase" system offered several advantages, reducing the usually required purification steps and opening a way to automatize the conjugation procedure.

Pharmacokinetic monitoring of mycophenolate mofetil in kidney transplanted patients.

Mycophenolate mofetil (MMF) is a new immunosuppressant drug used in association with cyclosporin and oral corticosteroids to prevent acute rejection following renal allograft transplantation. MMF is an ester pro-drug of mycophenolic acid (MFA), the true active species, into which it is completely transformed after oral administration. The recommended initial dose to prevent kidney transplant rejection is 2 g/day irrespective of body weight, 1 g twice daily. The goal of this study was to correlate dosage (fixed or by body weight) and toxic effects to some non-compartmental values such as peak level (Cmax), time to peak level (Tmax) and trough level (Cmin). In a small number of patients who had already reached the plasma steady state, we found a large inter-patient variability, while the same qualitative pharmacokinetic profile (as Tmax) was conserved. At plasma trough level > 4 microg/ml some serious toxic effects were observed, whereas at Cmin < 2 microg/ml, there were some cases of interstitial rejection. There was also a negative correlation between dosage and body weight, suggesting that dosages related to body weight might be better than fixed ones. Finally, monitoring plasma level of drug from transplantation to at least 12 months after surgery, at fixed MFA dosage, a small but significant decline of MFA plasma levels was found.