Frances W. J. Beck

Effect of adjuvant disease in rats on cyclophosphamide and isophosphamide metabolism

Abstract After the injection of a variety of arthritogenic adjuvants into male Wistar rats, hepatic activation of cyclophosphamide and isophosphamide is rapidly and profoundly depressed. This selective injury is largely reversible with phenobarbital and principally restricted to the liver microsomal protein fraction, which demethylates aminopyrine and N , N -dimethylaniline and generates “alkylating metabolites” from cyclophosphamide in vitro . Evidence is presented, based upon both metabolite excretion studies and the duration of hexabarbital-induced hypnosis, that this phenomenon is not an artifact in vitro and must be seriously considered in evaluating both the efficacy of potential anti-arthritic drugs against the rat adjuvant arthritis and their toxicity in these arthritic animals. A quantitative separation of two pathological responses to the same adjuvant may be obtained: (1) in Buffalo rats, whose liver metabolism may be profoundly impaired while they suffer minimal (or no) arthritis after being inoculated with adjuvants which are truly arthritogenic in other rat strains; (2) with Mycobacterium tuberculosis dispersed in methyl oleate, which induces minimal arthritis in Wistar rats, but nevertheless impairs their liver metabolism over a prolonged period (14 days or more). Drug metabolism appeared to be normal in rats with two other immunologically mediated “inflammatory diseases” (graft vs host disease and allergic encephalomyelitis) and in other rodents examined after adjuvant inoculations. A novel bioassay for cyclophosphamide and isophosphamide metabolites is described which utilizes their ability to prevent grafted rat lymphocytes from initiating a graft vs host reaction in tolerant recipient rats. At least four alkylating metabolites of cyclophosphamide were found in rat bile and tentatively identified by thin-layer chromatography. The possible error in relying on changes in urinary excretion (rather than biliary excretion) of drug metabolites as a guide to changes in hepatic xenobiotic metabolism is discussed.

Adjuvant disease in rats: biochemical criteria for distinguishing several phases of inflammation and arthritis.

Summary (1) The impaired drug metabolism, associated with adjuvant arthritis, spontaneously returns towards normal after 6 weeks. (2) The plasma protein thiol titer falls precipitiously with onset of arthritis but eventually returns to normal. (3) Based on these and other criteria, at least five phases of the disease can be distinguished which are summarized in Table III. (4) The Buffalo rat may develop massive arthritis, though relatively resistant to conventional adjuvants. Support by the USPHS (Grant No. GM 15759) and the V. A. is gratefully acknowledged.

Effect of cyclophosphamide on a local graft-versus-host reaction in the rat: Influence of sex, disease and different dosage regimens

A new pharmacological method for screening potential immunosuppressive drugs, the local graft-versus-host reaction in rats, has been used to evaluate the efficacy of cyclophosphamide applied at varous times in the development of this reaction.This drug was relatively ineffective when applied to the F1 hybrid recipient of the graft cells (splenic lymphocytes) prior to grafting, rather more effective when given only to the parental donor prior to harvesting the graft cells, and most effective when given to the recipient either immediately after the graft or after a delay of three days.Biliary and hepatic metabolites of cyclophosphamide diminished the competence of parental lymphocytes to evoke the graft response. By contrast, cyclophosphamide itself was devoid of such activity in vitro. Non-enzymic decomposition (hydroxylation) of cyclophosphamide with the Udenfriend system (Fe++, ascorbate, EDTA) efficiently generated in vitro graft-deactivating agents.Fortified liver preparations from normal female rats formed alkylating metabolites at a much slower rate, and adjuvant-arthritic male rats were less capable of generating graft deactivating cyclophosphamide metabolites in vitro, than liver preparations from normal male rats. However cyclophosphamide appeared to be no less effective in normal female or arthritic male rats in vivo, than in normal male rats, in controlling the graft-versus-host response. This lack of correlation between rates of hepatic cyclophosphamide metabolism and evident bioefficacy as an immunosuppressive drug is discussed, with special reference to similar findings by Sladek concerning rates of bioactivation and anti-tumor efficacy.

Impaired Drug Metabolism in Rats Associated with Acute Inflammation: A Possible Assay for Anti-injury Agents

Summary 1. Many acute irritants, e.g., turpentine, mimicked Freunds adjuvant (FA) in causing rapid depression of hexobarbital and cyclophosphamide metabolism in rats in vivo. 2. Metabolism of cyclophosphamide and aminopyrine in vitro by liver homogenates was often subnormal if liver donor animals had been acutely inflamed (rear paw, tail) 48 hr previously. 3. Three classes of irritants/inflammagenic stimuli were distinguished by their effects on liver drug metabolism. 4. Carrageenan as routinely used for anti-inflammatory screening had a peculiar, selective effect on drug metabolism. 5. Drug metabolism was distinctly subnormal in animals inoculated with FA (rear paw, tail) at all stages prior to developing arthritis. 6. Drug metabolism was almost normal in prearthritic animals when FA was inoculated ip in a front paw or ear. Ear inoculations induced arthritis consistently in Lewis and Wistar rats. 7. Several drugs which prevented development of arthritis failed to normalize drug metabolism in the 4-day period after inoculating FA in rear paw or tail. 8. 6-MP and dexamethasone prevented the acute depression of drug metabolism and may be prototypes of a new class of anti-injury agents. We are grateful to Dr. C. M. Pearson for continued encouragement, the USPHS (NIH Grant GM 15759) for financial support and to Mrs. D. J. Whitehouse for surgical assistance.

The local graft versus host reaction in the rat as a tool for drug mechanism studies

1 A local graft versus host reaction in rats permits the evaluation of drug action on (i) donor (parental) animals and (ii) recipient animals (F1 hybrid) prior to establishing a lymphoid cell graft to detect drugs which might attenuate immunocompetence over a prolonged period (e.g. steroids). 2 Treatment of the recipient animals after a lymphoid graft readily detects anti‐proliferative agents and may disclose other agents which interfere with the recognition of non‐self by viable graft cells. 3 Treatment of the cell graft in vitro before innoculation into the recipient animal permits the detection of alkylating agents, wihch effectively deactivate graft cells at non‐toxic concentrations. 4 Examples are given of how this reaction may be used to evaluate functionally the effects of chemical modification of the cell surfaces of lymphocytes and to detect active metabolites generated from a precursor drug (e.g. cyclophosphamide). 5 Some novel immunosuppressant drugs are described.

I. Drug Sensitivity of Rat Adjuvant Arthritis, Induced with ‘Adjuvants’ Containing no Mineral Oil Components

Summary 1. Adjuvant arthritis induced with mineral oil adjuvants was resistant to acetylsalicylic acid and flufenamic acid when given prophylactically, and only moderately sensitive to phenylbutazone. 2. Adjuvant arthritis induced in the same rat strain with 4 metabolizable adjuvants (M. tuberculosis with squalene or squalane or butyl palmi-tate or butyl stearate) was considerably more sensitive to acetylsalicylic acid, phenylbutazone and flufenamic acid. 3. These alternative adjuvants offer promise for screening weaker, aspirin-like (antiarthritic) drugs in rats, to derive less toxic (ulcero-genic, leukopenic) drugs for use in man. We are grateful to Dr. C. M. Pearson for continued encouragement, the USPHS (NIH grant GM 15759) for financial support, to Drs. R. Per-rin and J. K. Pollard, (Calbiochem) for gifts of butyl palmitate and commercial adjuvants (Per-rigens), and Mr. J. Fitzgerald, Miss R. Stremel and Mrs. E. Taylor for their unfailing technical assistance.

Some (pharmacological) properties of chloracetaldehyde, an oxidation product and potential metabolite of cyclophosphamide

Circumstantial evidence is presented that chloracetaldehyde is generated from cyclophosphamide by chemical oxidation in vitro with peroxide and might possibly be formed in vivo as a product of cyclophosphamide bioactivation.Chloracetaldehyde has several attributes of an agent capable of suppressing cellular immunity. Thus it prevents a graft-versus-host reaction (both in vivo and in vitro), is cytostatic and under certain conditions can abrogate an ongoing immunopathy (EAE).These properties are also exhibited by HN-2 and certain alkylating metabolites of cyclophosphamide.

Lymphocyte deactivation by (potential immunosuppressant) alkylating metabolites of cyclophosphamide

SummaryThe potential immunosuppressant activity of the following compounds derived from cyclophosphamide (CPA) was evaluated: 8 known metabolites, oxidation products and hydrolytic products of CPA containing the mustard moiety; the mixture of alkylating products formed from CPA by chemical and biological oxidation in vitro; acrolein. Mechlorethamine (HN2) and mannomustine were included in this survey as reference compounds.Drug action on lymphoid cells was assayed by ability to block/prevent 2 immunological diseases in rats (GvHR, EAE), GvHR in mice, and the mitogen response of human peripheral lymphocytes. These drug activities of the mustards and CPA derivatives are compared with their toxic/cytostatic and lipophilic properties.It is proposed that some of the more hydrophilic mustards and CPA metabolites may deactivate lymphocytes through an exoalkylating action upon the cell surface — possibly reacting with membrane-associated DNA —exemplifying the concept of a cell surface poison or ‘ZOG’.

Lymphocyte surface poisons: Disulfides and thiolsulfonates

Eight disulfides (I-VIII) and a thiolsulfonate (IX) were promising blocking agents of lymphocytes in graft-versus-host reactions (GvHR) without comensurate intracellular effects. The blocking effects were assayed through inhibition of the local GvHR after parental lymphocytes had been incubated with agents at suitable concentrations and then inoculated into F1 hybrid offspring. The intracellular effects were assessed beforehand by measuring the inhibition of [6-3H]thymidine incorporation by lymphocytes in the presence of a wide range of concentrations of agents. Concentration levels which induced no greater than approx. 50% inhibition of the [6-3H] thymidine incorporation were considered to reflect sufficiently small intracellular effects and were used for the subsequent GvHR comparisons. Cellular survival always was 90% or more for the GvHR tests (unless stated otherwise), even when inhibition of thymidine incorporation was as high as 50%; hence the thymidine data are useful not only as guides for dose levels in the GvHR but also as leads to new agents that may show immunosuppressive or anti-leukemic activity through intracellular effects. Structural specificity of the active compounds as cell-surface poisons is evidenced by little or no activity (less than 30% inhibition of GvHR) of 28 other disulfides, 2 trisulfides, 2 Bunte salts, and 8 other thiolsulfonates. Active agents may owe this function to replacement of the H of SH in cell-surface thiol receptors by an SR group. Glutathione did not significantly inactivate agents, probably because the products of reaction also are active disulfides. When two agents (III, IX) were given orally or intraperitoneally to F1 hybrid recipients of untreated parental cells, doses of 10--15 mg/kg produced a GvHR inhibition of 17--53%.

Queries about vaccines containing squalene.

‘Where there is much desire to learn there of necessity will be much arguing, much writing and many opinions: for opinion in good men is but knowledge in the making.’ John Milton 1644, English poet, in Areopagitica (a tract defending freedom of the Press). Squalene is again in the news. It is now being used widely as an adjuvant/immunostimulant to enhance vaccines against the ‘swine flu’ (A/H1N1) 2009. Hence, it is appropriate to briefly review its chequered history from at least three viewpoints (Table 1). This correspondence considers some of the early history of squalene (a polyunsaturated hydrocarbon) as an immunostimulant, and its recent/projected use as an adjuvant to amplify the potency of anthrax and influenza vaccines. Three pandemic vaccines against ‘swine flu’ (A/H1N1), incorporating squalene emulsions as adjuvants, have been approved for use in the European Union and People’s Republic of China. Clinical trials with two of these vaccines are under way in the European Union, Canada, the United States, Chile and Argentina. Should drug companies fail to produce enough swine flu antigen to fill existing orders for vaccine, US public health authorities have the option of stretching the supplies of non-adjuvanted vaccine by adding squalene adjuvants as a ‘dose-sparing agent.’ However, in the interests of long-term public health, further discussion is still needed about the acceptability of vaccines containing squalene-based adjuvants for mass vaccination programs. Glib assumptions are made about the supposed safety of squalene, because it is (i) endogenous, being an essential intermediate for sterol/steroid biogenesis in mammals; (ii) present in the sebum on skin; and (iii) sometimes ingested from the diet or nutriceutical supplements, apparently without harm. By contrast, parenterally administered squalene can induce chronic immunopathies in rats and in mice resembling autoimmune diseases in humans (rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus). By this criterion, exogenous squalene must be considered a potential pathogen/intoxicant. Other contentious issues should also be considered before generally accepting squalene as an adjuvant. They include (a) development of antibodies to squalene, (b) human immunopathies possibly associated with (experimental) anthrax vaccines and (c) likely non-enzymatic oxidative transformation of squalene to toxic metabolites.