Gary D. Gray

Immunosuppressive, antiviral and antitumor activities of cytarabine derivatives

Abstract Although cytarabine (cytosine arabinoside, ara -cytidine, Cytosar) is a potent immunosuppressant, antiviral and antitumor agent in animals and man, maximum inhibitory effects require the use of complex injection schedules. Previous reports have shown that good immunosuppressive and antitumor activities were attained with simple injection schedules using the 5′-adamantoate derivative. The current results show that a variety of 5′-acylates were equally as active as the 5′-adamantoate in suppressing immune responses in rodents (hemagglutinin formation in mice and hamsters, skin graft rejection in rats), and as antitumor agents in mice (L1210 leukemia). Similar results were attained in protecting mice from the lethal effects of intracranial herpes simplex infection, and in inhibiting DNA synthesis in phytohemagglutinin-stimulated human lymphocytes. The hypothesis for the enhanced potency of these newer derivatives was as follows: After injection of these insoluble derivatives, there is a finite time required for dispersion and solubilization. The freely circulating derivatives are resistant to deamination (and inactivation). After enzymatic hydrolysis to the free acid and cytarabine, the latter is then free to exert its inhibitory activities. The net effect is the maintenance of relatively low levels of cytarabine for long periods of time.

The demonstration of two γ-globulin subclasses in the goat

Abstract Two γ-globulin subclasses, designated γ G 1 and γ G 2 , have been identifed in goat serum. With similar sedimentation coefficients (6·4 S 20,ω ), goat γ G 1 and γ G 2 have both common and distinct antigenic determinants. The identification of the two subclasses was made possible by the use of rat and guinea pig antisera which reacted against both the common and individual determinants, in contrast to rabbit antisera, which detected only the common antigenic determinants. The existence of the two subclasses may be important in view of the wide-spread use and commercial availability of goat globulin preparations.

The immunosuppressive activity of ara-cytidine. I. Effects on antibody-forming cells and humoral antibody.

SUMMARY The immunosuppressive effects of ara-cytidine were studied on the cellular and humoral antibody response of mice. The immunosuppressive activity of ara-cytidine allowed dissection of the slow and rapid phases of antibody-forming cell (AFC) appearance. The cellular and humoral antibody responses both exhibited the same time of greatest sensitivity to ara-cytidine. The antibody-forming cell, hemolysin, and hemagglutinin responses were most susceptible to ara-cytidine inhibition 2 days after sheep red blood cell challenge. Injections of ara-cytidine had little effect except during the period of rapid AFC appearance. A normal but diminished primary antibody response occurred after ara-cytidine injections were stopped, except when multiple daily injections were continued for 11 days. In this case, no antibody could be detected after 22 days, and it was speculated that after 11 days insufficient amounts of antigen were present to initiate a detectable response. In contrast to the primary response, no delayed response was found during ara-cytidine inhibition of a secondary antibody response. The primary and secondary antibody forming cell responses were equally sensitive to ara-cytidine inhibition. IgM and IgG antibody-forming cells (detected -by the indirect plaque technique) were inhibited to the same extent by ara-cytidine injections. The immunosuppressive activity of ara-cytidine was dramatically increased by multiple daily injections which suggests that ara-cytidine has a very short immunosuppressive half-life in the mouse, and which corroborates previous reports describing increased anti-leukemic activity of ara-cytidine using multiple daily injections.

Immunosuppressive effects of adamantoyl cytarabine--i. Inhibition of hemagglutinin formation and graft versus host reactions in mice.

Abstract The immunosuppressive effects of adamantoyl cytarabine [1-β- D -arabinofuranosylcytosine-5′-(1-adamantanecarboxylate); AdOCA] have been examined and compared with the parent compound, cytarabine, in two mouse immunologie systems: the hemagglutinin response to sheep erythrocytes, and the graft versus host reaction. Previous reports by the authors have shown that cytarabine effects were of short duration and that multiple daily injections were necessary for maximum immunosuppression. In contrast, AdOCA demonstrated long-lasting imrnunosuppressive effects after one injection. Data accumulated in other experiments clearly demonstrate the greater immunosuppressive activity of AdOCA compared with cytarabine. This was true irrespective of the basis of comparison (single injections, daily injections, milligram or mole). Further, AdOCA immunosuppression was observed before, during, and after antigenic challenge, whereas cytarabine was shown previously to be most imrnunosuppressive 2 days after antigenic challenge.

Feline polymorphonuclear leukocytes respond chemotactically to leukotriene B4 and activated serum but not to F-Met-Leu-Phe.

The chemotactic response of feline polymorphonuclear leukocytes (PMNs) to three types of chemoattractants was studied. Feline PMNs responded to leukotriene B4 as well as to agarose-activated autologous and homologous serum. However, no response was obtained to N-formylmethionylleucylphenylalanine (FMLP), and four similar peptides that activate the FMLP receptor (N-formylnorleucylleucylphenylalanine, N-formylmethionylphenylalanine, methionylleucylphenylalanine, and pepstatin.) Thus, feline PMNs are similar to equine, porcine, bovine and canine PMNs which also do not respond chemotactically to these peptides.

The immunosuppressive activity of ara-cytidine ii. Effects on the graft-versus-host reaction.

SUMMARY The effects of ara-cytidine (ara-C) on the graft-versus-host reaction (GVHR) have been studied. The immunosuppressive activity of ara-C in the GVHR was extremely dependent upon timing of injection. Injections of ara-C on day 3 in the host undergoing a GVHR were most effective in inhibiting GVHR splenomegaly. This indicates that in the mouse strains employed (C57BLC3BF1), the donor cells were proliferating most rapidly on day 3. The immunocompetence of donor cells to elicit a GVHR was not affected by a 5-day course of ara-G injection. The data reported here and in a companion report suggest that (1) the immunosuppressive half-life of ara-C in the mouse is extremely short, (2) ara-C injections are immunosuppressive only during phases of immunocyte proliferation initiated by antigenic stimulation, and (3) ara-C would be very useful for dissection of immune responses into rapid and slow periods of immunocyte proliferation.

Biochemical and biological studies with tubercidin (7-deaza-adenosine), 7-deazainosine and certain nucleotide derivatives of tubercidin.

Various biological and biochemical properties of tubercidin (Tu), tubercidin-5′-phosphate (pTu), the methyl ester of tubercidin-5′-phosphate (MepTu), tubercidin-3′,5′-cyclic phosphate, several dinucleoside phosphates of tubercidin, and the chemical deamination product of Tu, 7-deazainosine (7-DI), including whole animal toxicity and drug distribution, were compared. All compounds containing Tu were much more cytotoxic to KB cells in culture than was 7-DI (ID50s 0.006-0.05 vs. 0.52 μg/ml) both in the resting and growing phases of cell metabolism. No evidence of conversion of MepTu to Tu or pTu was obtained after incubation in vitro with KB or red blood cells, whereas pTu was partially converted to Tu under the same conditions by KB cells. Tu and pTu were absorbed into and retained by blood cells when incubated in vitro (90–99% absorbed) to a much greater extent than were MepTu or 7-DI (5 and 60%). Tu inhibited DNA, RNA and protein synthesis by KB cells after short-term exposure to 4 or 20 μg/ml in vitro whereas MepTu showed very little effect on macromolecule synthesis even at 60 μg/ml under the same test conditions. Tu and pTu were quite inhibitory to Penicillium oxalicum in vitro whereas MepTu and 7-DI were inactive at identical concentrations. In mice, Tu and pTu were more toxic than MepTu or 7-DI, both on an acute and subacute basis. Both Tu and pTu were toxic orally whereas MepTu was not at the same dosage. While 7-DI showed very high serum (40 μg/ml) and urine levels (4000 μg/ml) after a single I.V. dose of 25 mg/kg in the dog, Tu showed low activity and recovery of biologically active material in the urine (0.25% for Tu, 25% for 7-DI). Considerably more tritiated material with biological activity was found in the urine of dogs which had received MepTu (25% of dose recovered as 3H, 13.7% as bioactivity). The mobilities on paper or silica gel of the excretion products in urine from dogs receiving Tu or MepTu differed, particularly on the bioautogram. Tu, MepTu and 7-DI were studied for their effects on hydrocortisone and tryptophan-induced tryptophan pyrrolase synthesis in rat liver. In these experiments, MepTu and 7-DI inhibited hydrocortisone induced enzyme synthesis at doses of 43 and 32 mg/kg, respectively (20 μmoles/rat). Tu was active at 16 mg/kg (10 μmoles/rat). Inhibitory activity was also observed when the compounds were administered 24 hr before steroid. At drug dosages that clearly inhibited the hydrocortisone induced stimulation, Tu and MepTu did not affect the tryptophan induced increase while 7-DI was inhibitory. The tubercidin derivatives did not stimulate aged TP preparations. Reactions of adenine or tubercidin derivatives with hematin, as determined by difference spectroscopy, were not consistent with TP stimulatory activities. These studies demonstrate several unique biological and biochemical effects for MepTu which could not have been predicted on the basis of its relatively simple chemical structure.

The immunosuppressive activity of ara-cytidine (cytarabine). 3. Effects of canine renal allograft rejection and hemagglutinin formation.

SUMMARY The effect of cytarabine (ara-cytidine, cytosine arabinoside), a potent antitumor and immunosuppressive agent, on canine renal transplantation has been studied. Cytarabine did not prolong the survival of the renal allografts although a modification of the histological pattern of rejection was observed. In contrast, the antibody (hemagglutinin) response induced by injections of sheep red blood cells was significantly suppressed by cytarabine. In addition, the appearance of both total and mercaptoethanol-resistant antibody titers were delayed. Cytarabine injections in normal and kidney transplant dogs resulted in a general leukopenia. No selectivity for leukocyte type was observed.

ARA-C AND DERIVATIVES AS EXAMPLES OF IMMUNOSUPPRESSIVE NUCLEOSIDE ANALOGS

Comprehensive discussions on the immunosuppressive activities of nucleoside analogs have been included in recent review a r t i ~ l e s . ~ . ’ ~ The purine nucleosides are being discussed in detail elsewhere in this annal. Since, in general, the pyrimidine nucleoside analogs display interesting immunosuppressive activities, it was decided to review one of these in detail. Ara-C (1-B-D-arabinofuranosylcytosine, cytarabine, Cytosar R )26 has been the pyrimidine nucleoside analog most exhaustively studied and offers a fair representation of most of the activities of classical nucleoside analogs.

Countergradients of Nonchemotactic Ligands for N-Formylmethionylleucylphenylalanine (FMLP) Receptors Promote FMLP-Induced Chemotaxis

Abstract Using the under-agarose chemotaxis assay, addition of compounds known to inhibit chemotaxis of leukocytes toward N-f-Met-Leu-Phe (FMLP) was made to the first well in a line of three wells, leukocytes to the middle well, and a slight excess of FMLP to the third well. The compounds included rifampin, fusidic acid, carbobenzoxy Phe-Met, phenylbutazone, sulfinpyrazone, sulfasalazine, and sulfapyridine. The countergradients created in this system markedly stimulated, not inhibited, locomotion toward FMLP. These results, based on functional responses, confirm data using radiolabeled FMLP and support the hypothesis that these compounds are nonchemotactic ligands for FMLP receptors.