Pulmonary delivery of nanoparticles of insoluble, iodinated CT X-ray contrast agents to lung draining lymph nodes in dogs.

Lung cancer continues to be a leading cause of death around the world. Staging of this disease is critically dependent upon the involvement or noninvolvement of the lymph nodes which drain the region of lung containing the lesion/tumor. Palpation, unenhanced CT, and lymph node excision (i.e., mediastinectomy) are currently used to ascertain the status of these regional draining lymph nodes. The work reported herein details the first efforts toward the pulmonary instillation of iodinated nanoparticles for contrast-enhanced CT of lung draining lymph nodes. The data reflect the impact of dose, time post instillation, and formulation (surfactant) upon the observed CT enhancement of the tracheobronchial lymph nodes of beagle dogs. In addition, initial safety is discussed with both macroscopic and microscopic observations. The results indicate that pulmonary instillation of small volumes of iodinated nanoparticles could be successfully used to aid staging of lung cancer by CT imaging.

Renal effects of iodixanol in experimental animals.

The effects of the new nonionic dimeric hexa-iodinated contrast media (CM) iodixanol on renal function and morphology were investigated in 7 independent studies in rats, rabbits and monkeys and compared with other iodinated CM. No significant effect on serum creatinine levels was seen at doses up to and including 5 g I/kg in rats and 10.5 g I/kg in rabbits. An immediate and transient increase in proteinuria was found in rabbits when 10.5 g I/kg was administered as a bolus, and when 12.5 g I/kg was administered as a slow infusion in a comparative study with several CM. Increased serum elimination half-life was shown by measuring serum iodine concentrations after the infusion of 12.5 g I/kg. The effect of a high dose of iodixanol on proteinuria and elimination half-life were in this study in the same range as those of the monomeric nonionic CM, but less pronounced than those of the monomeric ionic CM. Reduced renal capacity was induced in male rats by performing unilateral nephrectomy 4 weeks before i.v. injection of iodixanol or iopamidol (2g I/kg). The administration of CM did not affect renal function monitored as serum concentrations of creatinine and urea. The vacuolation of renal proximal tubular cells and kidney iodine retention were investigated in rats 48 hours after administration of different doses of iodixanol or iotrolan. The no-effect level for vacuolation was 0.5 g I/kg for both CM. Iodine retention was higher in male than females rats, and was higher for iodixanol than iotrolan at the 2 highest dose levels (3 and 5 g I/kg). No difference in iodine retention was found at the other dose levels (0.25-1g I/kg). The reversibility of renal proximal tubular vacuolation after administration of iodixanol was studied in male rats (1.2 g I/kg) and monkeys (1.2 and 3.6 g I/kg). The vacuolation was more pronounced in rats than in monkeys. Vacuolation was completely reversed in all rats 3 weeks after dosing, and 2 of 3 monkeys 3 days after a dose of 1.2 g I/kg. The degree of vacuolation evident in renal percutaneous biopsy specimens from monkeys 14 days after i.v. administration of iodixanol at a dose of 3.5 g I/kg was not significantly different to that in control animals. In conclusion, iodixanol affected renal function to the same degree as did the nonionic monomeric and dimeric comparative media, but to a lesser degree than the ionic monomers. The degree of renal proximal tubular cell vacuolation induced by iodixanol seems to be species-dependent, being less pronounced and more quickly reversed in monkeys than rats.

The integration of immunotoxicology in drug discovery and development: Investigative and in vitro possibilities.

The development of new pharmaceuticals requires a tremendous investment of time and resources. The early integration of investigative toxicology studies and new toxicological methods, such as immunotoxicological studies, into the selection of new drug candidates facilities compound safety evaluation, reduces risk, and improves development cycle time. Experience at Sterling Winthrop Inc. has led us to believe that immunotoxicology assessment represents an important and useful tool in drug discovery and development, but that it should not be applied as a routine screening procedure. Instead, immunotoxicity evaluation should be initiated as a response to knowledge about the class of drug under investigation, or when data from other studies suggest an effect on the immune system. Three examples of this approach are given and each one demonstrates the potential of in vitro immunotoxicology assays to reduce the time, resources and animal use required in the discovery and development of new drug candidates.

Clinical pathologic alterations associated with subcutaneous administration of recombinant human interleukin-4 to cynomolgus monkeys.

Recombinant human interleukin 4 (rhuIL-4) is a candidate for the treatment of refractory cancer based on its potential to enhance immune function. Recombinant human IL-4 was administered subcutaneously at 0, 1, 5, or 25 micrograms/kg/day for 28 days with a 14-day recovery to male and female cynomolgus monkeys as part of the preclinical safety evaluation. Clinical pathologic changes related to treatment with rhuIL-4 were evidence of consumptive coagulopathy, erythrocyte fragmentation, lymphocytosis, and lymphocyte morphologic changes indicative of marked antigenic or mitogenic stimulation, mild eosinophilia and neutrophilia, hypoalbuminemia, hypocholesterolemia, and hypertriglyceridemia. Based on data obtained after the 14-day recovery period, the clinical pathologic changes associated with rhuIL-4 administration were considered to be reversible.

Preclinical evaluation of recombinant human interleukin-4.

Recombinant human IL-4 (rhuIL-4) is primate-specific and produces multiple biologic effects on lymphoid cells involved in protection against cancer. RhuIL-4 was evaluated in the cynomolgus monkey to support clinical studies for the immunotherapy of cancer. Administration of rhuIL-4 to monkeys by SC injection of 0, 0.5, 2.5 or 12.5 micrograms/kg BID for one-month (with two-week recovery) resulted in alterations in clinical chemistry and hematology (CCH) parameters consistent with a consumptive coagulopathy. Histomorphologic evaluation revealed increased granulopoiesis, testicular atrophy, and proliferative and inflammatory vascular lesions (VL). IVL principally affected the arterial tree with some proliferation of medial smooth muscle. During the latter part of the treatment and recovery period. CCH parameters approached or returned to pretreatment values, the former finding attributed to the production of antibody to rhuIL-4. At final necropsy, bone marrow appeared normal, and IVL decreased in incidence and severity. ELISA studies of serum indicated 50-90% of the monkeys developed antibody titers > 1000 by Day 22 (not observed in man). The frequency and severity of adverse effects due to rhuIL-4 in the clinic appear to be does-related and reversible with few objective responses to therapy observed. Common toxicities included milk to moderated fever and fatigue and an occasional change in hematopoietic, hepatic and renal function. The monkey predicted hematologic findings, but not all target organ effects.

Pre-clinical toxicity of IL-4: a model for studying protein therapeutics.

The purpose of this presentation was to review issues and findings in the pre-clinical development and evaluation of recombinant human protein therapeutics. Since human cytokines and lymphokines are endogenous proteins, their pre-clinical development and evaluation would seem straightforward and their toxicities minimal. Unfortunately, the pre-clinical development of this class of agents has been problematic and confounding. Some of the clinical toxicities and pharmacodynamics have been predicted by the pre-clinical evaluation and others have not. Some molecules are species specific which limits species selection for pre-clinical evaluation. Other confounding issues include: route of exposure, synergy of toxicity with other lymphokines, length of study design, immunogenicity, predictiveness of pre-clinical evaluation and iatrogenic toxicities. An approach used by SWPRD in the evaluation of this class of molecules was discussed. Insight gained during the pre-clinical and clinical development of these molecules should simplify the further development of protein therapeutics that follow. Specific studies with recombinant human interleukin-4 (rhuIL-4) were reviewed in detail as part of a pre-clinical safety evaluation. Native IL-4 has properties that exemplify many of the immune recognition-induced lymphokines and is produced principally by activated T-lymphocytes CD4+. It is a co-factor in B-cell proliferation and enhances ex vivo B-cell expansion and is believed to be a candidate for the treatment of refractory cancer based on this immune enhancement ability. rhuIL-4 is a 15,400 molecular weight cytokine produced in a yeast expression system.(ABSTRACT TRUNCATED AT 250 WORDS)

Histomorphologic observations for cynomolgus monkeys after subchronic subcutaneous injection of recombinant human interleukin-4.

Recombinant human interleukin-4 (rhuIL-4) is a candidate for the treatment of refractory cancer based on its potential to enhance the function of the immune system. Total daily dosages of 0 (placebo control), 1, 5, or 25 micrograms/kg of rhuIL-4 were given as divided (b.i.d.) subcutaneous dosages to male and female cynomolgus monkeys (5/sex/group) for 1 month followed by a 2-week recovery. Histomorphologic evaluation of 3/sex/group at 1 month revealed vascular lesions, granulocytic hyperplasia, and seminiferous tubular atrophy attributed to treatment with rhuIL-4. Dosage-dependent proliferative and inflammatory vascular lesions with eosinophil infiltration affected principally the arterial tree. After 2 weeks of recovery, these lesions, including chronic endarteritis and chronic and/or obliterative arteritis, occurred with an overall lower incidence, and were not observed for monkeys from the 1.0 micrograms/kg/day group. Granulocytic hyperplasia in bone marrow observed for monkeys from all groups given rhuIL-4 at 1 month was not present after 2 weeks of recovery. Seminiferous tubular atrophy was observed for monkeys from the 5 and 25 micrograms/kg/day groups at 1 month and after 2 weeks of recovery.

Serum amyloid A protein concentration measured by radial immunodiffusion in Abyssinian and non-Abyssinian cats.

Serum amyloid A (SAA) protein concentration was determined by use of radial immunodiffusion (RID) in 4 groups of cats: Abyssinian cats with amyloidosis, healthy Abyssinian cats without clinical evidence of amyloidosis, hospitalized non-Abyssinian cats, and clinically normal non-Abyssinian cats. Mean SAA concentration in Abyssinian cats with amyloidosis was significantly (P = 0.05) higher than mean SAA concentration in healthy Abyssinian cats without clinical evidence of amyloidosis and in hospitalized non-Abyssinian cats. Mean SAA concentration in clinically normal non-Abyssinian cats was significantly (P = 0.05) lower than mean SAA concentration in healthy Abyssinian cats without clinical evidence of amyloidosis and in hospitalized non-Abyssinian cats. Affected and healthy Abyssinian cats, however, could not reliably be distinguished on the basis of SAA concentration, because of the wide range of SAA values in these 2 groups of cats.

Suppression of cytolytic function in murine lymphocytes exposed to 1,4-bis[(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD): inhibition of proliferation by cytotoxic T-lymphocyte precursors.

Previous reports have shown that exposure of murine splenic lymphocytes to the substituted anthraquinone 1,4-bis[(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD) results in a persistent, strong immunosuppression which is limited to induction of cytotoxic T-lymphocytes (CTL). In the present study the cellular mechanism of this specificity was examined in detail. Proliferation of T-lymphocytes is inhibited by in vitro exposure to AEAD, with suggestive evidence that this inhibition may be selective for the CTL precursor cells. Activation and differentiation of CTL precursors into functional CTL, as well as the function of T-helper lymphocytes, was unaffected by AEAD exposure. Rather, the committed CTL precursor cells are prevented from clonal proliferation, resulting in a much smaller population of antigen-induced CTL effectors. Finally, it was shown that AEAD is unable to prevent the anamnestic response of CTL memory cells to eliciting antigen. Taken together these data provide strong evidence that AEAD-induced CTL suppression results from its antiproliferative effect, directed primarily toward the CTL precursor subpopulation. This effect is manifested as decreased CTL function due to lower absolute number of specific CTL, since AEAD has no significant direct effect on expression of either specific or polyclonal cytolysis by T-lymphocytes.

Selective immunosuppression following exposure to a novel anthraquinone, 1,4-bis[(2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (AEAD).

The immune response of murine splenic lymphocytes was characterized following both in vivo and in vitro exposure to the novel and highly-substituted anthraquinone AEAD. Gross indicators of toxicity such as changes in body weight, lymphoid organ weights or lymphoid organ cellularity were unaffected. Similarly, the antibody plaque-forming cell response and natural killer cell function were unaltered. The most consistent effect noted was the selective suppression of the cytotoxic T-lymphocyte (CTL) response, which persisted for an extended period of time following in vivo exposure at concentrations of 0.75-3.0 micrograms/g. In contrast to the CTL suppression induced by the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]-anthracene, AEAD-induced CTL suppression could not be restored by addition of exogenous interleukin-2. Studies are currently underway to further characterize AEAD-induced immunosuppression at the cellular and molecular levels.

Evaluation of the immunotoxicity of beta-hexachlorocyclohexane (beta-HCH).

beta-HCH, an isomeric contaminant formed during the manufacture of the insecticide lindane, is a persistent environmental and food chain pollutant which has been reported to exhibit estrogenic activity in rodents and in fish. To investigate potential toxic effects on the reproductive and immune systems, beta-HCH was fed to female B6C3F1 mice for 30 days. Mice exposed to 0, 100, or 300 mg of beta-HCH/kg of diet were evaluated for changes in ovarian and uterine histology, body weight, lymphoid organ weight and histology, splenic cellularity, antigen-specific IgM and IgG plaque-forming cells (PFC), proliferative responses to mitogens, natural killer (NK) cell activity, and induction of cytolytic T lymphocytes. The ovaries and endometrial epithelium exhibited normal architecture. No alterations were observed in body weight, lymphoid organ weight and histology, or splenic cellularity whereas significant changes were found in several immune functions at the 300 mg/kg dose. Proliferation of splenocytes to the mitogens LPS, PHA, and Con A was decreased by 39, 43, and 57%, respectively. T-lymphocyte-mediated cytolysis of tumor targets was decreased by 25% with a concurrent reduction of 45% in NK activity. There was no significant reduction in the number of IgM or IgG PFC in exposed animals. These data indicate that beta-HCH causes nonestrogenic immune function changes in the adult mouse without gross changes in lymphoid organ weight, histology, or cellularity.

Comparison of a radioisotopic incorporation method and the mouse ear swelling test (MEST) for contact sensitivity to weak sensitizers.

A radioisotopic incorporation assay utilizing [125I]iododeoxyuridine was compared to the standard mouse ear swelling test (MEST) for the strong sensitizers dinitrofluorobenzene and oxazolone, and for the three weak sensitizers ethylenediamine (EDA), glutaraldehyde, and nickel sulfate. Mice were sensitized epicutaneously on the abdomen for 4 consecutive days prior to challenging the left ear with the test agent and the right ear with the vehicle. A comparison of the mean difference between the test and the control ears showed that measuring reactivity 48 hr postchallenge on Day 7 is the most sensitive time period in the radioisotopic incorporation method. Both the isotopic and MEST assays gave positive results with the potent sensitizers, although the response detected by isotopic labeling of emigrating cells was up to 1000-fold greater than that determined by ear swelling measurements. No response was detected to the moderate to weak sensitizer EDA in either assay. Reactivity to glutaraldehyde was not detected by the radioisotopic assay but was minimally responsive and significant by the MEST. The opposite was true for nickel sulfate where minimal but significant reactivity was seen in the isotopic assay but not in the MEST. Although the radioisotopic assay had the advantages of being more quantitative and of having improved sensitivity, it was of no greater value than the MEST for detecting weak sensitizers. It was concluded that the mouse was not a suitable model for routinely detecting reactivity to weak sensitizers regardless of which of the two assays were used.

Isolation and characterization of amyloid protein AA in the Abyssinian cat.

Amyloid fibrils were isolated by extraction in deionized water from the kidneys of an Abyssinian cat with familial renal amyloidosis. The fibrils were suspended in a buffer containing 6 M guanidine hydrochloride and reduced and alkylated using dithiothreitol and iodoacetid acid. The resulting amyloid fibril subunit protein was isolated by chromatography on a column of Sepharose CL6B. It was fragmented using cyanogen bromide, and the resultant peptides were separated by reverse phase high performance liquid chromatography. The protein was characterized by determination of the amino acid sequence of the cyanogen bromide fragments using a Beckman 890C sequencer. The primary structure of this amyloid fibril subunit protein showed strong homology with amyloid protein AA found in man and animals with spontaneous and experimentally induced reactive systemic amyloidosis. This study confirms the reactive nature of familial renal amyloidosis in the Abyssinian cat and suggests that this disease may be a valuable spontaneous animal model for the study of familial Mediterranean fever in man.

Adverse effect of exercise on immune complex-mediated glomerulonephritis.

To assess the effect of strenuous daily exercise on immune complex-mediated glomerulonephritis (GN), rabbits were randomly assigned to one of three experimental groups: Group I (n = 12): treadmill exercise for 28 days plus twice weekly intravenous injections of saline. Group II (n = 10): treadmill exercise for 28 days plus twice weekly intravenous bovine serum albumin (BSA) injection. Group III (n = 9): intravenous doses of BSA, as in group II, but no exercise. Blood and urine samples were collected from each animal periodically during the 28-day experimental period. On the 29th day of the study all animals were sacrificed and tissue taken for renal histopathologic studies. We found that in group II (exercise + GN) abnormal albuminuria was more frequent (p less than 0.001), blood urea nitrogen (BUN) levels rose significantly with time (p less than 0.02) and hematuria (blood in renal tubules) was more common (p less than 0.05), compared to group III (GN only). The differences between groups II and III could not be explained by the effect of exercise alone since group I (exercise only) developed no abnormal albuminuria, BUN levels or hematuria during the course of the study. These findings suggest that strenuous exercise superimposed on active immune complex-mediated GN results in worsening of the abnormal glomerular function.

Complement depletion accelerates the clearance of immune complexes from the circulation of primates.

Binding of immune complexes (IC) to erythrocytes in vitro is the result of interaction between C3b sites on the IC, and complement receptors type I (CRI) expressed on primate erythrocytes. Recent evidence indicates that primate erythrocytes can also rapidly bind large, preformed IC in vivo. This study was undertaken to determine if the binding of IC to baboon erythrocytes in vivo is complement dependent and to examine the effect of complement depletion on IC clearance from the circulation. The results indicate that complement depletion in vivo reduced the binding of IC to erythrocytes. There was relatively little binding of IC to leukocytes in both the complement-depleted and complement-repleted condition. Thus, the majority of IC not bound to erythrocytes remained free in the plasma and, consequently, IC infusion during the complement-depleted state resulted in increased plasma IC concentrations. This was associated with a rapid disappearance of IC from the circulation. By contrast, in the normal or complement-repleted state, a large fraction of the IC became bound to erythrocytes during IC infusion, which resulted in lower plasma IC concentrations. Under these conditions, a more gradual rate of disappearance of IC from the circulation was observed. The relatively abrupt clearance of IC from the circulation in the complement-depleted state could not be accounted for by increased hepatic or splenic uptake. These data indicate that, in contrast to previous studies in nonprimates, complement depletion in primates results in accelerated removal of IC from the circulation. This suggests that factors such as hypocomplementemia and deficient expression of erythrocyte CRI, which are known to occur in certain IC-mediated diseases, may promote IC uptake by organs vulnerable to IC-mediated injury.

Factors influencing the binding of large immune complexes to the primate erythrocyte CR1 receptor.

This study was undertaken to ascertain the relationship between the size of immune complexes and their capacity to be bound by primate erythrocytes, and to examine the role of complement in the binding of very large immune complexes to erythrocytes. Preformed bovine serum albumin-rabbit anti-bovine serum albumin immune complexes were solubilized in a fivefold antigen excess, and the supernatant fluids were subjected to differential centrifugation to select immune complexes of varying size. The size profile of immune complexes was measured on isokinetic sucrose gradients. Immune complexes were incubated with erythrocytes, and unbound immune complexes were separated from erythrocyte-bound immune complexes by centrifugation on Percoll. The results indicate that large immune complexes were bound more efficiently by primate erythrocytes than were smaller immune complexes. Depletion of complement by a variety of procedures abrogated binding of immune complexes to erythrocytes. Only the erythrocytes of primates had the capacity to bind immune complexes. Baboon or rabbit sera supported immune complex binding to baboon or human erythrocytes. In contrast, human and guinea pig sera supported immune complex binding to human erythrocytes, but these sera failed to support immune complex binding to baboon erythrocytes. These data indicate that the immune complex size and the source of serum complement are important factors which influence the binding of immune complexes to primate erythrocytes.

Primate erythrocyte-immune complex-clearing mechanism.

Previous in vitro studies have shown that immune complexes (IC) that fix complement can bind to the C3b receptor on primate erythrocytes. The in vivo function of this erythrocyte receptor, however, is unknown. This study was undertaken to determine whether the binding of IC to erythrocytes in vivo might play a role in the removal of IC from the circulation. Baboons and rhesus monkeys were prepared with a catheter in the ascending aorta to infuse IC and in the abdominal aorta, renal, hepatic, and portal veins to monitor changes in binding and clearance of IC across kidney, liver, and spleen + gut, respectively. Autologous 51Cr-labeled erythrocytes were infused intravenously and allowed to equilibrate. Preformed IC (125I-labeled bovine serum albumin [BSA] rabbit anti-BSA) were then infused into the ascending aorta at a constant rate for 120 s. Blood samples were drawn at frequent intervals for 30 min from all catheters below the IC injection site. Each blood sample was then centrifuged on percoll to separate IC bound to erythrocytes from IC in plasma or bound to buffy coat cells. This resulted in an "erythrocyte fraction" beneath the percoll that contained the IC bound to erythrocytes, and a "plasma/buffy coat fraction" above the percoll that contained the IC in plasma and IC bound to buffy coat cells. Analysis of these data showed that the majority of the IC infused into the circulation rapidly became bound to erythrocytes. However, by 5 min after beginning the IC infusion, most of this IC load had been removed from the erythrocytes as they traversed the liver. In contrast, IC on erythrocytes did not deposit in kidney. The IC-bearing erythrocytes themselves were not trapped or detained by any organ. IC in the plasma/buffy coat fraction of blood were removed from the circulation but at a relatively low rate and almost entirely by the liver. These findings suggest that primate erythrocytes intercept large complement-fixing IC in the circulation causing the IC to adhere to the erythrocyte until th e IC-bearing erythrocyte traverses liver where the IC is deposited, and the erythrocyte is returned to the circulation. This primate erythrocyte-IC-clearing mechanism may be important in the protection against diseases mediated by deposition of circulating IC.

Effect of moderate daily exercise on acute glomerulonephritis.

Exercise can induce proteinuria, hematuria and cylindruria in normal individuals. This suggests that exercise adversely affects glomerular function. In this study we examined the impact of moderate daily treadmill exercise on the glomerulonephritis (GN) of 'one-shot' bovine serum albumin (250 mg/kg i.v.) serum sickness in rabbits. We found that exercise alone increased serum creatinine concentration (Scr) but exercise plus GN did not increase Scr further. Blood urea nitrogen values were unchanged. Albuminuria and the renal histopathology findings were not different between the exercised and non-exercised groups of rabbits. Muscle cytochrome oxidase and mitochondrial protein concentrations were not increased in the exercised animals. We conclude that exercise, below the level that causes exercise adaptation in muscle enzymes, does not adversely affect this form of acute GN.

Immunotherapy of a murine sarcoma using specific antitumor immune RNA.

The effect of IRNA therapy in a polyoma-induced fibrosarcoma of C3H mice has been studied. Although a protocol that transferred in vitro cytotoxicity was used, no reduction in incidence of tumors, tumor size, or survival was demonstrated. In one experiment, enhancement of tumor growth occurred. It is concluded that cytotoxicity does not reflect in vivo changes and that therapeutic trials of IRNA are premature.