Carlos L. Krumdieck

Supplementation with folic acid during methotrexate therapy for rheumatoid arthritis. A double-blind, placebo-controlled trial.

The folic acid antagonist methotrexate (n-10-methyl-aminopterin) is useful in low doses (2.5 to 20 mg/wk) for treating chronic inflammatory diseases [1-7]. Many trials have established the efficacy of methotrexate in rheumatoid arthritis [7-13]. Compared with other disease-modifying drugs, methotrexate has the highest probability of drug continuation at 10 years. Dose response-related toxic effects have been reported in 30% to 90% of patients given methotrexate [13]. Toxic effects include gastrointestinal intolerance, hematologic abnormalities, alopecia, hepatotoxicity, and pulmonary toxicity [14-22]. Some side effects of methotrexate administration, such as gastrointestinal intolerance, mimic complicated folate deficiency [23]. Folate deficiency occurs frequently in patients with rheumatoid arthritis; further, folate stores are decreased in patients with rheumatoid arthritis who take methotrexate, suggesting that impaired folate status is related to toxicity [24-26]. Folic acid supplementation has been reported anecdotally to lessen toxicity in patients receiving methotrexate treatment [27, 28]. In a 6-month, double-blind, placebo-controlled trial, 7 mg of folic acid weekly (1 mg/d or 2265 nmol/d) decreased methotrexate toxicity without affecting efficacy [29]. This was confirmed by Stewart and colleagues [30] in a retrospective chart review. Folinic acid (leucovorin, citrovorum factor) is a one-carbon-substituted, fully reduced folate that has also been administered during methotrexate therapy [31-36]. Low doses of the vitamin (1 to 7 mg/wk) have decreased methotrexate toxicity [35, 36]. Higher doses negate efficacy and lessen toxicity [31, 32]. Thus, the folinic acid dose may critically affect the efficacy of methotrexate therapy. The influence of the folic acid dose on methotrexate toxicity and efficacy remains controversial, and the effects of different doses of folic acid are not known [37, 38]. Some investigators argue that if toxic effects occur, the most rational approach is to reduce the dose of methotrexate rather than to provide folic acid supplements [37]. We designed a larger and longer study to evaluate different doses of folic acid, assuming that toxicity could be reduced without changing the efficacy of methotrexate. Methods Participants Patients aged 19 to 78 years who fulfilled the American College of Rheumatologys revised criteria for rheumatoid arthritis consented to participate in the trial [39]. Enrollment criteria included rheumatoid arthritis diagnosed more than 6 months previously, onset after the age of 16 years, and at least three of the following signs or symptoms: 3 or more swollen joints, 6 or more tender joints, at least 45 minutes of morning stiffness, and a Westergren erythrocyte sedimentation rate 28 mm/h. Referring rheumatologists and the principal investigator did the screening. Exclusion criteria included serious concomitant medical illnesses, liver enzyme levels twice the upper limit of normal, leukocyte counts less than 3.5 109/L or platelet counts less than 150 109/L, and use of methotrexate within the past 6 months. Gold salts were stopped for at least 10 days before the trial. This short washout period mirrors actual rheumatology practice. Patients remained under the care of their rheumatologists, abstained from alcohol use, did not become pregnant, and received stable doses of aspirin and nonsteroidal anti-inflammatory drugs. If prednisone was taken at entry, the dose could not exceed 10 mg/d. Hydroxychloroquine therapy was allowed during the study. Study Design Figure 1 shows the trial design. To maintain the double-blind status of the trial, the statistician carried out the randomization using a computer program in which the algorithm was transparent and a coded vial number represented the treatment assignment. Patients were assigned to treatment groups by a sequential treatment assignment process designed to balance the sample with respect to baseline features, including age, sex, folate-containing vitamin use, rheumatoid factor status, and prednisone use [40]. Patients agreed to discontinue therapy with folate-containing vitamins during the trial. Rheumatoid factor was considered positive if the level was more than 30 IU/mL or if the titer was more than 1:160. Patients received either visually identical placebo or 5 mg (low-dose folic acid group) or 27.5 mg folic acid (high-dose folic acid group) each week, prepared by the Hospital Investigational Drug Service. Spectrophotometric analysis indicated that the mean SD folic acid content was 1 0.15 mg (2.3 0.3 mol) and 5.5 0.3 mg (12.5 0.7 mol) per capsule in the low-dose and high-dose folic acid groups, respectively. Lederle Laboratories provided the methotrexate (Rheumatrex; Pearl River, New York), which was started in a median oral dose of 16.5 mol (7.5 mg) per week and increased in 5.5-mol (2.5 mg) increments at the rheumatologists discretion. Methotrexate was taken either in an undivided or a divided dose (that is, every 12 hours for three doses). The methotrexate dosing regimens were identical among the study groups. Folic acid supplements were given 5 days per week when methotrexate was not ingested. Compliance with the regimen was reinforced using a digital reminder cap (Counter Cap; Senetics, Boulder, Colorado). All participants and investigators were blinded to vitamin capsule content until the study was complete. Figure 1. Study design for the double-blind, placebo-controlled trial. Clinical Assessment Patients were evaluated immediately before methotrexate initiation at a mean of 13, 26, 39, and 53 weeks (Figure 1). Each patient was assessed by the same physician-nutritionist (SLM). Two research assistants (JSA or WHV) did the joint evaluations. In most cases, patients were examined by the same observer throughout the study. The joint counts for tenderness and swelling were not significantly different between the two observers (P = 0.6 for tenderness; P = 0.9 for swelling). The following variables were evaluated at each visit: 1) number of the 58 diarthrodial joints with swelling; 2) number of the 60 joints with tenderness on pressure or with pain on passive motion (or both); 3) joint swelling and tenderness indices, expressed as a sum, with each joint graded for swelling as 0 (none), 1 (mild), 2 (moderate), or 3 [severe]; 4) mean grip strength (three measurements) for both hands expressed in mm Hg; 5) duration of morning stiffness expressed in hours; 6) patients and physicians global assessments of disease activity graded as 0 (asymptomatic), 1 (mild), 2 (moderate), 3 (severe), or 4 (very severe); 7) current medications and doses; 8) a 1-day dietary recall using the Minnesota Nutrition Data System software, Food Database version 6A, Nutrient Database version F21 [41]; 9) eight activities of daily living assessed and averaged at baseline and at 12 months using the modified Health Assessment Questionnaire and scored on a scale of 1 (no difficulty) to 4 (unable to perform) [42]; and 10) presence, duration, intensity, and severity of toxic effects at every follow-up visit and every 3 weeks by telephone interview (done by SLM). Laboratory Assessments At the initial visit, complete blood cell count, Westergren erythrocyte sedimentation rate, liver enzyme (aspartate amino transferase and alkaline phosphatase), rheumatoid factor by nephelometry, and serum creatinine values were obtained. The complete blood cell count, creatinine, and liver function tests were repeated at all visits. If laboratory values were obtained more frequently than stipulated in the protocol, they were recorded and became part of the toxicity score. Blood for plasma and erythrocyte folate levels, using a methotrexate-resistant Lactobacillus casei microbiological assay, was drawn at all visits [43]. At baseline, blood was drawn for a vitamin panel (plasma and erythrocyte folate, vitamins B12, A, C, and E, carotene, riboflavin, thiamin, pyridoxine) [44-51]. If patients had abnormal values for any of the vitamins, other than folate, the abnormality was treated with single vitamin supplements. Radiographic Assessment Hand and wrist radiographs taken within 6 months of entering the trial were assessed by one of the rheumatologists (GSA) without knowledge of study status. Joint erosions and space narrowing were determined by the modified method of Sharp [52]. Results were expressed as the mean raw scores for joint erosion and joint space narrowing. Toxicity: Frequency and Severity Toxic effects and discontinuation of therapy with study medications due to toxicity were considered primary outcomes. We determined a toxicity score, modified from the one previously used, for each patient at each visit or until methotrexate therapy was discontinued [29] (Appendix). Efficacy We determined patient response to treatment using a modification of the criteria used in our previous folic acid supplementation trial and by others [8, 29, 53]. We defined marked improvement in the joint swelling index and the joint tenderness and pain index as a net decrease of 50% or more in value at any follow-up visit compared with visit 1. We defined moderate improvement as a 30% to 49% net decrease in the index, and no change as the index value remaining within 30% of the original value. We defined worsening as an increase in the index value of 30% or more. We examined the effects of folic acid supplementation on changes in physician and patient global assessments, grip strength, morning stiffness, erythrocyte sedimentation rate, findings of the modified Health Assessment Questionnaire, complete blood cell counts, blood folate levels, and dietary folate and vitamin B12 intake. Table 1. Demographic and Selected Clinical Characteristics of 79 Patients with Rheumatoid Arthritis* Statistical Analysis Sample Size and Analysis In our previous study, the mean toxicity scores for patients receiving low-dose folic acid and placebo recipients were 0.21 and 1.06, respectively, for

A new instrument for the rapid preparation of tissue slices

Abstract A manually operated microtome for the rapid preparation of live tissue slices is described. The instrument operates submerged in an isotonic solution and incorporates an automatic mechanism for the removal of the cut slices which are gently carried by a stream of fluid to a strainer reservoir. An inexperienced operator can obtain circular slices of nearly identical thickness at a rate of one every 3 or 4 s. Mechanical trauma to the tissues is minimized and the temperature and oxygenation of the medium are easily controlled.

Polygammaglutamyl metabolites of methotrexate

Abstract Heretofore unrecognized metabolites of methotrexate (MTX) have been detected in human red blood cells and isolated from rat liver and viscera. The metabolites from the rat were identified as 2,4-diamino-N 10 -methylpteroylglutamyl-γ-glutamic acid [MTX(G 1 )] and 2,4-diamino-N 10 -methylpteroylglutamyl-γ-glutamyl-γ-glutamic acid [MTX(G 2 )] by comparison with authentic synthetic compounds.

Role of Vitamin B12 and Folate Deficiencies in Carcinogenesis

A significant body of experimental evidence supports the notion that a deficiency of either vitamin B12 or folic acid enhances the activity of various carcinogens. Unifying mechanisms are proposed to explain this cocarcinogenic role.

Dynamic organ culture of precision liver slices for in vitro toxicology

The lack of a reproducible method for the production of thin tissue slices has hindered the use of liver slices as an in vitro tool for hepatotoxicity studies. Fresh human, rat, and rabbit liver was processed using a mechanical slicer. With this instrument, precision (5% of thickness) liver slices in the submillimeter range could be produced at a rapid rate. Slices were prepared from fresh livers in chilled, oxygenated buffer to minimize trauma. Following incubation for up to 20 h in a dynamic organ culture system, histology of incubated slices suggested that 250 m precision-cut slices were optimum in regard to morphology relative to liver slices incubated under conventional organ culture conditions. Addition of bromobenzene to the culture showed time-dependent hepatotoxicity based on two classic parameters of cell degeneration. Histological evidence is presented which suggests the usefulness of this system for hepatotoxicity studies and the production of focal necrosis in vitro.

Osteopontin, a substrate for transglutaminase and factor XIII activity.

Osteopontin (OPN), an extracellular matrix cell adhesion protein, was found to serve as a substrate for the incorporation of radiolabelled putrescine mediated by a commercial preparation of guinea pig liver transglutaminase. Preliminary evidence also suggests that OPN serves as a substrate for the plasma transglutaminase, Factor XIIIa. While the protein substrates to which OPN is linked in vivo have not been identified, it is reasonable to speculate that this capacity of OPN may dictate its extracellular location and thereby affect its role in bone homeostasis, tumorigenesis, metastasis, resistance to bacterial infections or, perhaps, wound repair.

In vitro cytotoxicity of allyl alcohol and bromobenzene in a novel organ culture system

Two well-known hepatotoxicants, allyl alcohol (AA) and bromobenzene (BB), were studied using an in vitro system of cultured liver slices from control and phenobarbital-treated rats, respectively. Dose- and time-dependent increases in media lactate dehydrogenase (LDH), and decreases in slice K+ content and in protein synthesis were observed in rat liver slices incubated with either compound at concentrations between 0.1 and 1 mM over a period of 6 hr. The histopathological changes which occurred in the intoxicated slices appeared to parallel these biochemical changes. Additionally, the toxicity of either BB or AA, evaluated at 4 hr, was inhibited when slices were preincubated for 30 min with beta-ethyl-2,2-diphenylvalerate hydrochloride (SKF 525-A) (0.1 mM) or pyrazole (1.0 mM), respectively. In this in vitro incubation system the cytotoxicity of xenobiotics can be studied under conditions where the multicellular hepatic lobular architecture is partially maintained, and alterations in biochemical and functional processes may be correlated to pathological changes.

A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity

Purpose: Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver. Experimental Design: The Krumdieck thin-slice tissue culture system was used to obtain and culture slices of tumor xenografts of ovarian cancer cell lines, human primary ovarian tumors, and human liver. We determined the viability of slices in culture over a period of 36 to 48 hours by ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl-2-(4-sulfophenyl)-2H-tetrazolium, inner salt)]) (MTS) assay. In vitro Hey cells, slices of Hey xenografts, and human ovarian tumor or human liver slices were infected with 500vp/cell of either replication competent wild-type adenovirus (Ad5/3wt), conditionally replicative adenovirus (Ad5/3cox-2), or the replication deficient adenovirus (Ad5/3luc1). At 12-, 24-, and 36-hour intervals, the replication of adenoviruses in these slices was determined by quantitative reverse transcription-PCR of adenoviral E4 copy number. Results: Primary tumor slices were able to maintain viability for up to 48 hours after infection with nonreplicative virus (Ad5luc1). Infection of Hey xenografts with Ad5/3cox-2 showed replication consistent with that seen in Hey cells infected in an in vitro setting. Primary tumor slices showed replication of both Ad5/3wt and Ad5/3cox over a 36-hour time period. Human liver slices showed replication of Ad5/3wt but a relative reduction in replication of Ad5/3cox-2 indicative of conditional replication “liver off” phenotype, thus predicting lower toxicity. Conclusions: The thin-slice model system represents a stringent method of ex vivo evaluation of novel replicative adenoviral vectors and allows assessment of human liver replication relative to human tumor replication. This is the first study to incorporate this system for evaluation of therapeutic efficacy and replicative specificity of conditionally replicative adenoviruses. Also, the study is the first to provide a valid means for preclinical assay of potential conditionally replicative adenovirus-based hepatotoxicities, thus providing a powerful tool to determine therapeutic index for clinical translation of conditionally replicative adenoviruses.

The oxidative cleavage of folates: A critical study

Abstract Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the C9N10 bond to produce the corresponding p-aminobenzoyl-polyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkalinc (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, one-carbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and N5-formyl-tetrahydrofolic acid are cleaved at the C9N10 bond to produce p-aminobenzoylglutamic acid. N5, N10-methenyl-tetrahydrofolic acid, N5,N10-methylene-tetrahydrofolic acid, and N10-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N10-formyl-folic acid which is completely stable to the oxidative treatment employed. N5-methyl-tetrahydrofolic acid is not cleaved either but is oxidized to N5-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The γ-glutamyl peptide linkage is completely stable to oxidation. Using p-amino-[3,5-3H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance of these findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2-14C]folic acid to label the folates in vivo is presented.

Mechanisms of Homocysteine Toxicity on Connective Tissues: Implications for the Morbidity of Aging

It is proposed that chronic moderate hyperhomocysteinemia has a causal role in a number of common diseases of late life, including occlusive vascular disease, cognitive decline, senile osteoporosis and presbyopia. These diseases are seen as clinical counterparts of the main manifestations of homocystinuria (vascular occlusions of arteries and veins, mental retardation, osteoporosis and ectopia lentis, respectively) that develop only after many years of exposure to moderately elevated homocysteine (Hcy) levels. The multisystem toxicity of Hcy is attributed to its spontaneous chemical reaction with many biologically important molecules, primarily proteins. The formation of these Hcy-adducts is dependent on time and Hcy concentration and leads to loss or diminution of function of the derivatized molecules. Irreversible homocysteinylation of long-lived proteins should lead to cumulative damage and progressive clinical manifestations. Fibrillin 1 is seen as the paradigm of extracellular connective tissue proteins that are specially susceptible to Hcy (and presumably Hcy thiolactone) attack. The prominent presence of epidermal growth factor (EGF)-like domains in fibrillin and in many other extracellular proteins of the coagulation, anticoagulation, and lipoprotein transport pathways, all of which malfunction in hyperhomocysteinemia, suggests that EGF-like domains may be preferential sites of homocysteinylation.