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A Multicentered Study of Lysine Therapy in Herpes simplex Infection

A Multicentered Study of Lysine Therapy in Herpes simplex Infection Dermatologica 156: 257-267 (1978) A Multicentered Study of Lysine Therapy in Herpes simplex Infection R ic h a r d S. G r if f it h , A r t h u r L. N o r in s and C h r is t o p h e r K a g a n Lilly Laboratory for Clinical Research, Wishard Memorial Hospital, Department o f Dermatology, University Hospital, Indiana University School of Medicine, Indianapolis, Ind., and Department of Pediatrics, Cedars-Sinai Medical Center, UCLA Center for the Health Sciences, Los Angeles, Calif. Key Words. Herpes simplex • Lysine therapy Abstract. Lysine appears to suppress the clinical manifestations o f herpesvirus in­ fection. 45 patients with frequently recurring herpes infection were given 312-1,200 mg of lysine daily in single or multiple doses. The clinical results demonstrated a beneficial effect from supplementary lysine in accelerating recovery from herpes simplex infection and suppressing recurrence. Tissue culture studies have demonstrated an enhancing effect on viral replication when the amino acid ratio of arginine to lysine favors arginine. The oppo­ site, preponderance of lysine to arginine, suppresses viral replication and inhibits cyto- pathogenicity of herpes simplex virus. The codons characterizing herpes simplex DNA apparently specify production of viral capsids at the expense of host cell histones. Introduction It has been estimated that 50-70% of the adult population of the world are afflicted with painful cutaneous lesions due to herpes simplex virus (HSV, type 1) [23], In recent years, a venereally transmitted HSV, type II, has also been recognized. These viruses may also cause encephalitis and have been linked epidemiologically to cervical cancer [7], Multiple recurrence of the cutaneous lesions is due to the herpesvirus becoming latent after a primary infection, usually during childhood, and then being repeatedly activated by subsequent provocations [1]. Although Received: August 23, 1977; accepted: September 5, 1977. 258 G r iffith /N o rin s/ K agan recurrent herpes simplex virus infections are usually self-limited, frequently the lesions are painful, persistent, or embarassing enough to warrant therapy. A variety of therapeutic measures have been disappointing. It has been shown through tissue culture that the infectivity of the virus is removed by dissolving the lipid coat in various solvents. Ether applied topically has been used in the treatment of labial herpes [20]. The cowpox virus used in the vaccination for smallpox for many years has had questionable acceptance as a vaccine for herpes infections [31]. Various dyes, neutral red and phenolsulfonphthalein, that have been shown to aid in photoinactivation of herpes simplex in vitro, have failed to show demonstrable efficacy when tried clinically with adequate controls [19], Analogs of pyrimidines and purines have been used topically to hasten heal­ ing of recurrent cutaneous herpetic lesions [10, 11], In severe herpes simplex infection in the immunosuppressed patient, adenine arabinoside has been used parenterally [3]. Evidence that the virus is found in a latent form in sensory neuron cells emphasizes that topical therapy may only be palliative [5], The systemic use of nucleoside analogs has recognized risks [3], Obviously, a nontoxic viral suppressive agent that could penetrate into viral-infested cells would be welcomed clinically. In an in vitro study, T ankersley [28] found that arginine encouraged whereas lysine antagonized herpesvirus replication and cytopathogenicity. K agan [8] observed clini­ cally the efficacy of lysine, suggested by T ankersley’s in vitro work. Patients and Methods All patients were volunteers who sought treatment for lesions due to recurrent herpes simplex infection. Subjects with sporadic manifestations were not included. There were 11 males and 34 females, aged 4-60 years. L-Lysine monohydrochloride was purchased through commercial channels (General Nutrition Corp., Pittsburgh, Penn.) in capsules or as tablets containing 100 or 312 mg of L-lysine. Therapeutic dosage, during an overt infection, was 800-1,000 mg daily. The maintenance dose for most patients was usually 312-500 mg daily. Though the patients were asked to take the medication continuously, exhaustion of supply or forgetfulness permitted evaluation of the effect of interrupted treatment. No attem pt was made to markedly alter dietary intake of natural foods. It was recom­ mended, however, that those foods known to be high in arginine (nuts, seeds, and choco­ late) be curtailed. A Multicentered Study of Lysine Therapy in Herpes simplex Infection 259 Results The 45 patients admitted to the multicenter study were followed for from 2 months to 3 years (table I) by three investigators. Since most of the subjects in the study were either hospital or industrial employees, only 3 cases were lost to follow-up. No adverse reactions were reported. There was an unex­ plained preponderance of females over males. No sex-related differences in therapeutic results were seen. Each patient was carefully questioned regard­ ing severity and duration of each episode and response to the administration of lysine compared to previous therapy. The pain disappeared abruptly overnight in virtually every instance, new vesicles failed to appear, and resolution in the majority was considered to be more rapid than in their past experience. The initiating lesion remained confined to one area in those patients who characteristically had experienced extension and progression to multiple sites. 2 patients who had previously Table I. Results of lysine treatment of herpes simplex infection Patient Age/sex Site of infection Culture Dosage, Duration Results Comment mg of Investigator follow-up Mild 39/M lip ND 400 daily 3 years suppressed stress related PH/G R A/G 53/M lip ND 400 daily 1 year suppressed sunlight PW/G 29/F lip HSV-I 400 b.i.d. 3 months suppressed lip HSV-II 624 b.i.d. 3 months SL/G 31/F suppressed 39/F lip ND 400 daily 3 months suppressed DT./G CT/K 12/M ND 312 daily1 2 months suppressed lip Moderate 26/F lower facial HSV-I 312 b.i.d. 3 years suppressed TS/G 28/F lower facial Neg 400 daily2 1 year failure PS/G RW/G 60/F mouth3 and lower face ND 400 b.i.d. 6 months suppressed 30/F lip and mouth ND 400 b. i.d. 6 months suppressed JW/G I6/F mouth ND 312 b.i.d. 3 years cured due to dental SS/S prosthesis ND 400 daily 4 months decreased JE/G 30/F lip 55/F lip ND 400 b.i.d. 2 years suppressed stress related EM /G HSV-I SN/G 29/F lips and mouth 624 daily 3 months suppressed ND 624 daily 3 months suppressed Tl/G 23/F lips, nose, chin 260 G r iffith /N o rins/ K agan Table / (Continuation) Patient Comment Agc/sex Site o f infection Culture Dosage, Duration Results Investigator mg of follow-up LB/G 28/F left forearm HSV-II 624 daily 3 months suppressed MS/G 24/F lips, nose, mouth, eyelid ND 624 daily 4 months suppressed RG/G 25/F lips and chin ND 3 months suppressed 1,200 daily FC/G 31/M lips ND 624 daily 3 months suppressed HP/G 47/F lips and mouth ND 624 daily 3 months suppressed MJ/G 53/F lips, nose, mouth HSV-I 312 daily 1 year suppressed JF/G 30/F lips, chin HSV-I 400 daily 6 months suppressed TW/N 4 /F lip ND 500 daily 1 year suppressed NQ/N 24/F ND 1,000 daily 9 months suppressed lip NP/N 37/F lip ND 1,000 daily 6 months suppressed TS/N 8/F lip ND 500 daily2 3 6 months decreased AG/N 16/F lip ND 1,000 daily 6 months suppressed BW/N 29/F buttocks ND 500 daily 8 months failure DC/N 31/M penis ND 1,000 daily 4 months suppressed JP/N 31/F lip ND 1.000 daily 1 year suppressed HT/K 8/M lip ND 312 daily1 2 months suppressed BB/K 9/M cheek and lip ND 312 daily1 1 month suppressed ER/K 10/F mouth ND 312 daily1 6 months suppressed lymphocytic leukemia RR/K l l / F mouth ND 312 daily1 6 months suppressed 12/F BF/K genital ND 312 daily1 6 months suppressed RF/K 14/M lip and mouth ND 312 daily1 6 months suppressed JN/K 18/F lip and vulva ND 312 daily1 6 months suppressed SA/K. 20/F lip and chin ND 312 daily1 6 months suppressed MY/K 20/M ND 312 daily1 6 months suppressed lip Severe CR/G 26/M conjunctiva and lower HSV-I 312 b.i.d. 2 years suppressed eyelid RN/N 3 8/F lip ND 1,000 daily 6 months suppressed DA/N 8/M around eye ND 500 daily 6 months suppressed SV/N 5/F lip; around eye ND 500 daily 1 year suppressed Incapacitating SW/G 29/F lower facial HSV-I 312 b.i.d. 3 years suppressed sunlight JW/G 26/F lower face and mouth ND 400 b.i.d. 3 months suppressed 1 Treated only during infection. 2 Higher dosage not tried. 3 Mouth mucous membrane. A Multicentered Study of Lysine Therapy in Herpes simplex Infection 261 lost work because of severe, incapacitating progressive herpes infection of the mouth and lips, had rapid control of their initial lesion and obtained continued suppression while on lysine. The most encouraging finding, other than more rapid control and limit­ ing of the infection, was the reduction in frequency of recurrences. Patients who sought help because of the persistence and frequency of infection were maintained infection-free while on lysine but found that within 1-4 weeks after stopping medication, return of the lesions could be predicted. For this reason, results were interpreted as suppressive rather than as a cure. Patients trying to establish a maintenance control dose would ‘break through' when the amount of lysine ingested was cut back too much. Main­ tenance dosage varied with the individual, but it became obvious that no Fig. I. Patient SWs extensive lesions, from which HSV-type 1 was cultured, failed to respond to topical therapy and had shown progression for four weeks prior to her clinic visit (a). Gradual improvement was observed on one 312 mg capsule of lysine twice daily for 1 month (b). A suppressive dose of 3 12 mg daily has been successful for the past 2 years. 262 G r iffith /N o rins/ K agan recurrence occurred in virtually all of the patients on 500 mg or more per day. Interestingly, most patients subject to frequent herpes infection had learned that a stinging pain was indicative that a new lesion was starting. In some instances a palpable nodule followed rapidly (hours). Taking thera­ peutic doses of 500-1,000 mg or increasing the maintenance dose would invariably abort recurrence. 3 patients associated occurrence and exacerbation of their herpetic lesions with sunlight. 2 not only responded to lysine therapy but were able to play tennis or vacation in Florida without any problems. The third was notable in that her skin disease did not respond at all to lysine. Herpesvirus could not be isolated from her lesions. Figure 1 illustrates an example of improvement related to lysine therapy. Discussion There is a theoretical rationale for the effectiveness of lysine in herpes simplex infection. The protein fraction I of the histone layer around the DNA of the eukaryotic or host cell has 28% lysine and only 3-4% arginine [2, 18]. The protein in the capsid coat around the DNA core o f the virus is the reverse; it has more arginine than lysine [9]. The preferred utilization of arginine to lysine in the protein being synthesized by the herpes-infested cell has been explained on the basis of the difference between the nucleoside composition of the cellular DNA and the viral DNA [9, 27]. There is support for the concept that the lysine-rich histones in the eukaryo­ tic cell are important in the maintenance of the structural integrity of the nuclear DNA [12, 17]. The herpesvirus uses the cytosine and guanine (CG) doublet containing codons for specification of amino acids which the mammalian cell utilizes only rarely. This specification can be illustrated by considering the amino acid arginine. The known code lists six arginine codons: CGU, CGC, CGA, CGG, AGA, and AGG. Since mammalian host cells have relatively low CG doublet frequency, they use the first four codons infrequently and rely mainly on AGA and AGG, whereas the herpesvirus makes frequent use of the CG-containing codons [29]. The above six codons can program for arginine whereas only two, AAA and AAG, program for lysine [14]. A shift A Multicentered Study of Lysine Therapy in Herpes simplex Infection 263 from the lysine codon AAG by only one nucleotide would program for arginine AGA (AAG, AAG, AAG->A, AGA, AGA, AG). Virus-specific protein is made quite rapidly by modifying the host cell translation apparatus. New arginyl tRNAs calling for arginine-rich proteins have been identified in the viral-infested host cell [26, 27], The results of the in vitro tissue culture studies substantiate the DNA ratio predicting lysine and arginine utilization. T ankersley [28] observed that maximal herpesvirus yield required an optimal pool o f amino acids. Using the plaque-forming method in his studies, complete Eagle’s medium had a virus yield of 4.5 (log PFU). Omitting a specific amino acid from the medium reduced the plaque formation. In the absence of arginine, plaque formation decreased to < 2 .0 . However, the reverse was true when lysine was left out of the medium: plaque formation increased rather than decreased; the virus yield in the absence of lysine increased to 4.75. Lysine added to the medium had an inhibitory action on viral replication (table II) [28], Plaque formation was 10 or less in arginine-free Eagle’s medium containing 70-280 p-g/ml of lysine. In Eagle’s medium containing arginine, 70 [i.g/ml of lysine showed 1,080,000 plaque-forming units (PFU). Four times the amount, 280 ¡jtg/ml, decreased plaque formation by 1 log, 110,000 PFU. Lysine antagonized the arginine effect on virus replication in tissue culture. It is of historical interest that in 1952 P earson et al. [22] while studying the propagation of the encephalomyelitis virus in tissue culture, found that lysine was the most inhibitory of the naturally-occurring amino acids [22], Levo-rotary lysine isomers were effective substitutes whereas the dextro- rotary forms were inert, suggesting an allosteric mechanism [22]. Table II. Lysine antagonism o f arginine effect on herpes simplex virus multiplication in tissue culture [28] Lysine concentrations, ug/ml Medium 70 180 280 Arginine-free Eagle’s medium 10' 10 10 Eagle's medium (with arginine) 1,080,000 190,000 110,000 1 Virus yield in plaque-forming units. 264 G r iffith /N o rins/ K agan K aplan el al. [9] performed even more definitive studies showing the change in amino acid utilization when tissue culture cells were infected with herpesvirus. Using ‘tagged' amino acids he found that during the 3- to 4-hour period before cytopathogenicity became apparent, the infected cell machinery, under the influence of the viral DNA, had shifted to making proteins with a preponderance of arginine over lysine. Arginine at the peak of its incorporation into proteins was 125% of the control while lysine utilization dropped to less than 40%. He concluded that his results showing the change in arginine utilization to lysine utilization was a switchover in the manufacture of cell-specific proteins to virus-specific proteins. This verified the shift, or switchover, of codon doublets brought about by the presence of the viral DNA CG content as suggested by Subak-Sharpe et al. [27], The rapid ‘take-over’ of the herpesvirus suggests that arginine might act as an operon coordinate inducer. A preponderance of lysine over arginine could either act as an allosteric enzyme inhibitor [4] or an operon coordinate repressor [15] of the capsid protein synthesis. The studies by T ankersley [28] showing that the lysine must be incor­ porated in the medium before inoculation to prevent viral replication and our clinical studies showing relapse when lysine is discontinued, emphasize that the effect of lysine is only suppressive, not curative. This phenomenon could explain the intermittent characteristic of herpes infection. Table III. Comparison of lysine-arginine in food (g/100 g of food)1 Arginine Lysine Milk 0.12 0.24 Meat 1.2 1.6 Eggs 0.7 0.7 3.7 Brewer’s yeast 2.1 Potatoes 0.1 0.2 Peas 1.6 1.5 Cereal2 0.6 0.4 Peanuts 7.4 2.4 Chocolate 4.0 2.0 1 Adapted from data in reference 6. 2 Nontoasted. A Multiccntcred Study of Lysine Therapy in Herpes simplex Infection 265 The diet of man contains L-lysine and L-arginine. Certain foods are low in lysine but have a relatively high arginine content; others are low in arginine (table III) [6]. Lysine can be synthesized by yeast cells. However, tissues of man apparently cannot synthesize lysine. Once lysine has lost its nitrogen, it cannot be reaminated. Man is totally dependent on his diet as a source of lysine [13]. Variations in absorption and excretion of lysine have been reported [21, 24, 25, 30]. Improper food selection may make adequate lysine intake precarious for some individuals [16]. The ingestion of cereals, seeds, nuts, chocolate, etc., should produce a high arginine to lysine ratio and favor herpetic lesions similar to the results obtained by adding arginine to the medium in the laboratory to induce herpes proliferation. C ontrari­ wise, the avoidance of these foods, coupled with the selection of foods with adequate lysine, such as dairy products, yeast, etc., should discourage herpes infection. This may explain the low incidence of herpes in infants prior to weening from a predominantly milk diet. Our studies indicate that relatively low doses of lysine added to that in the diet can offset the arginine effect. It is interesting that it has been shown since 1952 that lysine has a deterrent effect on viral replication in tissue cultures and since 1964 that arginine has an enhancing effect. Yet there were no clinical studies until K agan [8] reported in 1974 that lysine suppressed herpesvirus infections in patients. For several years we evaluated a herpes simplex vaccine that makes it possible to historically compare the relative merits of lysine therapy. Our results are so encouraging that more elaborate controlled studies, including the pharmacology of lysine, are now underway. References 1 Bud d in g h. C.J.; Schrum. D .I.; L andier. J.C., and G uidry, D.J.: Studies of the natural history of herpes simplex infections. Pediatrics, Springfield //.- 595-610 (1973). 2 B usch, H.: Histones and other nuclear proteins, p. 45 (Academic Press, New York 1965). 3 C h' ien, L.T.; C annon, N .J.; C haramella, L.J.; Dismukes, W.E.; W hitley, R.J.; B uchanan, R.A., and A lford, C.A., j r . : Effect of adenine arabinoside on severe herpesvirus hominis infection in man. J. infect. Dis. 128: 658-663 (1973). 4 Coh en, G .H .: Ribonucleotide reductase activity of synchronized KB cells infected with herpes simplex virus. J. Virol. 9: 408-418 (1972). 5 Editorial: Herpes virus. Latency in neural cells shown in animal models. Hospital Pract. 7: 32 (1972). 266 G r iffith /N o rin s/ K aoan 6 Heinz Nutritional Data; 6th ed. (Heinz International Research Center, Pittsburgh 1972). 7 J aw etz, E.; M elnick, J.L., and A delberg, E.A.: Review of medical microbiology; I Ith cd., pp. 436-437 (Lange Medical Publications, Los Altos 1974). 8 K agan, C. : Lysine therapy for herpes simplex. Lancet i: 137 (1974). 9 K aplan, A.S. ; Shimono, H., and Ben-Porat, T. ; Synthesis of proteins in cells infect­ ed with herpesvirus. III. Relative amino acid content of various proteins formed after infection. Virology 40: 90-101 (1970). 10 K aufman, H.E. : Clinical cure of herpes simplex keratitis by 5-iodo-2'deoxy-uridine. Proc. Soc. exp. Biol. Med. 109: 251 252 (1962). 11 K aufman, H.E.; E llison, E.D., and Tow nsend, W.M.: The chemotherapy of herpes iritis with adenine arabinoside and cytarabine. Archs Ophthal., N.Y. 84: 783-787 (1970). 12 Kinkade, J.M ., jr. : Differences in the quantitative distribution o f lysine-rich histones in neoplastic and normal tissues. Proc. Soc. exp. Biol. Med. 137: 113 1 — 1134 (1971). 13 K leiner, LS. and O rten, J.M .: Biochemistry; 6th ed., p. 484 (Mosby, St. Louis 1962). 14 Lehninger, A.L. : Biochemistry, the molecular basis of cell structure and function, p. 718 (Worth Publishers, New York 1970). 15 L ehninger, A.L. : Biochemistry, the molecular basis o f cell structure and function, p. 734 (Worth Publishers, New York 1970). 16 M ertz, E.T.: Genetic improvement of cereals. Nutr. Rev. 32: 129-131 (1974). 17 M irsky, A.E.; Bur dick, C.J.; D avidson, E.H., and Lillau, V.C.: The role of lysine- rich histones in the maintenance of chromatin structure in metaphase chromosomes. Proc. natn. Acad. Sci. USA 61: 592 597 (1968). 18 M urray, K.: The heterogenicity of histones; in Bonnf.r and Ts'o The nucleohistones, p. 24 (Holden-Day, San Francisco 1964). 19 M yers, M .G.; D aman, N.M .; C lark, J.E., and A rndt, K.A.: Failure of neutral red photodynamic inactivation in recurrent herpes simplex virus infections. New Engl. J. Med. 293: 945-949 (1975). 20 N ugent, G.R. and C hou, S.M.: Treatment of labial herpes. J. Am. med. Ass. 224: 132 (1973). 21 O mura, K.: Lysine malabsorption syndrome. A new type of transport defect. Pedia­ trics, Springfield 57: 102-105 (1976). 22 Pearson, H.E. ; L agerborg, D.L., and W inzler, R.J.: Effect of certain amino acids and related compounds on propagation of mouse encephalomyelitis virus. Proc. Soc. exp. Biol. Med. 70: 409-411 (1952). 23 Sabin, A.B. : Misery o f recurrent herpes. What to do? New Engl. J. Med. 293: 986-988 (1975). 24 Sh ih, V.E.; G lick, T.H., and Bercu, B.B.: Lysine metabolism in Reye's syndrome. Lancet ii: 163-164 (1974). 25 Simell, O.; Perheentupa, J.; R apola, J.; Visakorpi, J.K., and Eskelin, L.-E.: Lysinuric protein intolerance. Am. J. Med. 59: 229-240 (1975). 26 Subak-S harpe, H. and H ay, J.: An animal virus with DNA of high guanine + cyto­ sine content which codes for s-RNA. J. molec. Biol. !2: 924-928 (1965). 27 S ubak-S harpe, H .; Burk, R.R.; C rawford, L.V.; Morrison, J.M ., an d H ay, J.: An ap p ro ach to evolutionary relationships o f m am m alian DNA viruses through A Multicentered Study of Lysine Therapy in Herpes simplex Infection 267 analyses of the pattern of nearest neighbor base sequences. Quant. Biol. 31: 737-748 (1966). 28 T ankkrsley, R.W., j r . : Amino acid requirements of herpes simplex virus in human cells. J. Bact. 87: 609-613 (1964). 29 T r o pin, J.A.; R ottman, F.M .; Brimacombe, R.L.C.; L eder, P.; Bernfield, M.R., and N irenberg, M.W.: RNA codewords and protein synthesis. VI. On the nucleotide sequences of degenerate codeword sets for isolcucine, tyrosine, asparagine, and lysine. Proc. natn. Acad. Sci. 58: 807-811 (1965). 30 Van G elderen, H.H. and T eijema, H .L .: Hyperlysinaemia. Harmless inborn error of metabolism? Archs Dis. Childh. 48: 892-895 (1973). 31 W agner, R .R .: Herpes simplex: in Beeson and M c D ermott Textbook of medicine, pp. 117-119 (Saunders, Philadelphia 1967). R.S. G riffith, MD, Lilly Research Laboratories, PO Box6 18, Indianapolis, I N 46206(USA) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Dermatology Karger

A Multicentered Study of Lysine Therapy in Herpes simplex Infection

Griffith, Richard S.; Norins, Arthur L.; Kagan, Christopher
Dermatology , Volume 156 (5): 11 – Jan 1, 1978
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Karger
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© 1978 S. Karger AG, Basel
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1018-8665
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1421-9832
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10.1159/000250926
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Abstract

Dermatologica 156: 257-267 (1978) A Multicentered Study of Lysine Therapy in Herpes simplex Infection R ic h a r d S. G r if f it h , A r t h u r L. N o r in s and C h r is t o p h e r K a g a n Lilly Laboratory for Clinical Research, Wishard Memorial Hospital, Department o f Dermatology, University Hospital, Indiana University School of Medicine, Indianapolis, Ind., and Department of Pediatrics, Cedars-Sinai Medical Center, UCLA Center for the Health Sciences, Los Angeles, Calif. Key Words. Herpes simplex • Lysine therapy Abstract. Lysine appears to suppress the clinical manifestations o f herpesvirus in­ fection. 45 patients with frequently recurring herpes infection were given 312-1,200 mg of lysine daily in single or multiple doses. The clinical results demonstrated a beneficial effect from supplementary lysine in accelerating recovery from herpes simplex infection and suppressing recurrence. Tissue culture studies have demonstrated an enhancing effect on viral replication when the amino acid ratio of arginine to lysine favors arginine. The oppo­ site, preponderance of lysine to arginine, suppresses viral replication and inhibits cyto- pathogenicity of herpes simplex virus. The codons characterizing herpes simplex DNA apparently specify production of viral capsids at the expense of host cell histones. Introduction It has been estimated that 50-70% of the adult population of the world are afflicted with painful cutaneous lesions due to herpes simplex virus (HSV, type 1) [23], In recent years, a venereally transmitted HSV, type II, has also been recognized. These viruses may also cause encephalitis and have been linked epidemiologically to cervical cancer [7], Multiple recurrence of the cutaneous lesions is due to the herpesvirus becoming latent after a primary infection, usually during childhood, and then being repeatedly activated by subsequent provocations [1]. Although Received: August 23, 1977; accepted: September 5, 1977. 258 G r iffith /N o rin s/ K agan recurrent herpes simplex virus infections are usually self-limited, frequently the lesions are painful, persistent, or embarassing enough to warrant therapy. A variety of therapeutic measures have been disappointing. It has been shown through tissue culture that the infectivity of the virus is removed by dissolving the lipid coat in various solvents. Ether applied topically has been used in the treatment of labial herpes [20]. The cowpox virus used in the vaccination for smallpox for many years has had questionable acceptance as a vaccine for herpes infections [31]. Various dyes, neutral red and phenolsulfonphthalein, that have been shown to aid in photoinactivation of herpes simplex in vitro, have failed to show demonstrable efficacy when tried clinically with adequate controls [19], Analogs of pyrimidines and purines have been used topically to hasten heal­ ing of recurrent cutaneous herpetic lesions [10, 11], In severe herpes simplex infection in the immunosuppressed patient, adenine arabinoside has been used parenterally [3]. Evidence that the virus is found in a latent form in sensory neuron cells emphasizes that topical therapy may only be palliative [5], The systemic use of nucleoside analogs has recognized risks [3], Obviously, a nontoxic viral suppressive agent that could penetrate into viral-infested cells would be welcomed clinically. In an in vitro study, T ankersley [28] found that arginine encouraged whereas lysine antagonized herpesvirus replication and cytopathogenicity. K agan [8] observed clini­ cally the efficacy of lysine, suggested by T ankersley’s in vitro work. Patients and Methods All patients were volunteers who sought treatment for lesions due to recurrent herpes simplex infection. Subjects with sporadic manifestations were not included. There were 11 males and 34 females, aged 4-60 years. L-Lysine monohydrochloride was purchased through commercial channels (General Nutrition Corp., Pittsburgh, Penn.) in capsules or as tablets containing 100 or 312 mg of L-lysine. Therapeutic dosage, during an overt infection, was 800-1,000 mg daily. The maintenance dose for most patients was usually 312-500 mg daily. Though the patients were asked to take the medication continuously, exhaustion of supply or forgetfulness permitted evaluation of the effect of interrupted treatment. No attem pt was made to markedly alter dietary intake of natural foods. It was recom­ mended, however, that those foods known to be high in arginine (nuts, seeds, and choco­ late) be curtailed. A Multicentered Study of Lysine Therapy in Herpes simplex Infection 259 Results The 45 patients admitted to the multicenter study were followed for from 2 months to 3 years (table I) by three investigators. Since most of the subjects in the study were either hospital or industrial employees, only 3 cases were lost to follow-up. No adverse reactions were reported. There was an unex­ plained preponderance of females over males. No sex-related differences in therapeutic results were seen. Each patient was carefully questioned regard­ ing severity and duration of each episode and response to the administration of lysine compared to previous therapy. The pain disappeared abruptly overnight in virtually every instance, new vesicles failed to appear, and resolution in the majority was considered to be more rapid than in their past experience. The initiating lesion remained confined to one area in those patients who characteristically had experienced extension and progression to multiple sites. 2 patients who had previously Table I. Results of lysine treatment of herpes simplex infection Patient Age/sex Site of infection Culture Dosage, Duration Results Comment mg of Investigator follow-up Mild 39/M lip ND 400 daily 3 years suppressed stress related PH/G R A/G 53/M lip ND 400 daily 1 year suppressed sunlight PW/G 29/F lip HSV-I 400 b.i.d. 3 months suppressed lip HSV-II 624 b.i.d. 3 months SL/G 31/F suppressed 39/F lip ND 400 daily 3 months suppressed DT./G CT/K 12/M ND 312 daily1 2 months suppressed lip Moderate 26/F lower facial HSV-I 312 b.i.d. 3 years suppressed TS/G 28/F lower facial Neg 400 daily2 1 year failure PS/G RW/G 60/F mouth3 and lower face ND 400 b.i.d. 6 months suppressed 30/F lip and mouth ND 400 b. i.d. 6 months suppressed JW/G I6/F mouth ND 312 b.i.d. 3 years cured due to dental SS/S prosthesis ND 400 daily 4 months decreased JE/G 30/F lip 55/F lip ND 400 b.i.d. 2 years suppressed stress related EM /G HSV-I SN/G 29/F lips and mouth 624 daily 3 months suppressed ND 624 daily 3 months suppressed Tl/G 23/F lips, nose, chin 260 G r iffith /N o rins/ K agan Table / (Continuation) Patient Comment Agc/sex Site o f infection Culture Dosage, Duration Results Investigator mg of follow-up LB/G 28/F left forearm HSV-II 624 daily 3 months suppressed MS/G 24/F lips, nose, mouth, eyelid ND 624 daily 4 months suppressed RG/G 25/F lips and chin ND 3 months suppressed 1,200 daily FC/G 31/M lips ND 624 daily 3 months suppressed HP/G 47/F lips and mouth ND 624 daily 3 months suppressed MJ/G 53/F lips, nose, mouth HSV-I 312 daily 1 year suppressed JF/G 30/F lips, chin HSV-I 400 daily 6 months suppressed TW/N 4 /F lip ND 500 daily 1 year suppressed NQ/N 24/F ND 1,000 daily 9 months suppressed lip NP/N 37/F lip ND 1,000 daily 6 months suppressed TS/N 8/F lip ND 500 daily2 3 6 months decreased AG/N 16/F lip ND 1,000 daily 6 months suppressed BW/N 29/F buttocks ND 500 daily 8 months failure DC/N 31/M penis ND 1,000 daily 4 months suppressed JP/N 31/F lip ND 1.000 daily 1 year suppressed HT/K 8/M lip ND 312 daily1 2 months suppressed BB/K 9/M cheek and lip ND 312 daily1 1 month suppressed ER/K 10/F mouth ND 312 daily1 6 months suppressed lymphocytic leukemia RR/K l l / F mouth ND 312 daily1 6 months suppressed 12/F BF/K genital ND 312 daily1 6 months suppressed RF/K 14/M lip and mouth ND 312 daily1 6 months suppressed JN/K 18/F lip and vulva ND 312 daily1 6 months suppressed SA/K. 20/F lip and chin ND 312 daily1 6 months suppressed MY/K 20/M ND 312 daily1 6 months suppressed lip Severe CR/G 26/M conjunctiva and lower HSV-I 312 b.i.d. 2 years suppressed eyelid RN/N 3 8/F lip ND 1,000 daily 6 months suppressed DA/N 8/M around eye ND 500 daily 6 months suppressed SV/N 5/F lip; around eye ND 500 daily 1 year suppressed Incapacitating SW/G 29/F lower facial HSV-I 312 b.i.d. 3 years suppressed sunlight JW/G 26/F lower face and mouth ND 400 b.i.d. 3 months suppressed 1 Treated only during infection. 2 Higher dosage not tried. 3 Mouth mucous membrane. A Multicentered Study of Lysine Therapy in Herpes simplex Infection 261 lost work because of severe, incapacitating progressive herpes infection of the mouth and lips, had rapid control of their initial lesion and obtained continued suppression while on lysine. The most encouraging finding, other than more rapid control and limit­ ing of the infection, was the reduction in frequency of recurrences. Patients who sought help because of the persistence and frequency of infection were maintained infection-free while on lysine but found that within 1-4 weeks after stopping medication, return of the lesions could be predicted. For this reason, results were interpreted as suppressive rather than as a cure. Patients trying to establish a maintenance control dose would ‘break through' when the amount of lysine ingested was cut back too much. Main­ tenance dosage varied with the individual, but it became obvious that no Fig. I. Patient SWs extensive lesions, from which HSV-type 1 was cultured, failed to respond to topical therapy and had shown progression for four weeks prior to her clinic visit (a). Gradual improvement was observed on one 312 mg capsule of lysine twice daily for 1 month (b). A suppressive dose of 3 12 mg daily has been successful for the past 2 years. 262 G r iffith /N o rins/ K agan recurrence occurred in virtually all of the patients on 500 mg or more per day. Interestingly, most patients subject to frequent herpes infection had learned that a stinging pain was indicative that a new lesion was starting. In some instances a palpable nodule followed rapidly (hours). Taking thera­ peutic doses of 500-1,000 mg or increasing the maintenance dose would invariably abort recurrence. 3 patients associated occurrence and exacerbation of their herpetic lesions with sunlight. 2 not only responded to lysine therapy but were able to play tennis or vacation in Florida without any problems. The third was notable in that her skin disease did not respond at all to lysine. Herpesvirus could not be isolated from her lesions. Figure 1 illustrates an example of improvement related to lysine therapy. Discussion There is a theoretical rationale for the effectiveness of lysine in herpes simplex infection. The protein fraction I of the histone layer around the DNA of the eukaryotic or host cell has 28% lysine and only 3-4% arginine [2, 18]. The protein in the capsid coat around the DNA core o f the virus is the reverse; it has more arginine than lysine [9]. The preferred utilization of arginine to lysine in the protein being synthesized by the herpes-infested cell has been explained on the basis of the difference between the nucleoside composition of the cellular DNA and the viral DNA [9, 27]. There is support for the concept that the lysine-rich histones in the eukaryo­ tic cell are important in the maintenance of the structural integrity of the nuclear DNA [12, 17]. The herpesvirus uses the cytosine and guanine (CG) doublet containing codons for specification of amino acids which the mammalian cell utilizes only rarely. This specification can be illustrated by considering the amino acid arginine. The known code lists six arginine codons: CGU, CGC, CGA, CGG, AGA, and AGG. Since mammalian host cells have relatively low CG doublet frequency, they use the first four codons infrequently and rely mainly on AGA and AGG, whereas the herpesvirus makes frequent use of the CG-containing codons [29]. The above six codons can program for arginine whereas only two, AAA and AAG, program for lysine [14]. A shift A Multicentered Study of Lysine Therapy in Herpes simplex Infection 263 from the lysine codon AAG by only one nucleotide would program for arginine AGA (AAG, AAG, AAG->A, AGA, AGA, AG). Virus-specific protein is made quite rapidly by modifying the host cell translation apparatus. New arginyl tRNAs calling for arginine-rich proteins have been identified in the viral-infested host cell [26, 27], The results of the in vitro tissue culture studies substantiate the DNA ratio predicting lysine and arginine utilization. T ankersley [28] observed that maximal herpesvirus yield required an optimal pool o f amino acids. Using the plaque-forming method in his studies, complete Eagle’s medium had a virus yield of 4.5 (log PFU). Omitting a specific amino acid from the medium reduced the plaque formation. In the absence of arginine, plaque formation decreased to < 2 .0 . However, the reverse was true when lysine was left out of the medium: plaque formation increased rather than decreased; the virus yield in the absence of lysine increased to 4.75. Lysine added to the medium had an inhibitory action on viral replication (table II) [28], Plaque formation was 10 or less in arginine-free Eagle’s medium containing 70-280 p-g/ml of lysine. In Eagle’s medium containing arginine, 70 [i.g/ml of lysine showed 1,080,000 plaque-forming units (PFU). Four times the amount, 280 ¡jtg/ml, decreased plaque formation by 1 log, 110,000 PFU. Lysine antagonized the arginine effect on virus replication in tissue culture. It is of historical interest that in 1952 P earson et al. [22] while studying the propagation of the encephalomyelitis virus in tissue culture, found that lysine was the most inhibitory of the naturally-occurring amino acids [22], Levo-rotary lysine isomers were effective substitutes whereas the dextro- rotary forms were inert, suggesting an allosteric mechanism [22]. Table II. Lysine antagonism o f arginine effect on herpes simplex virus multiplication in tissue culture [28] Lysine concentrations, ug/ml Medium 70 180 280 Arginine-free Eagle’s medium 10' 10 10 Eagle's medium (with arginine) 1,080,000 190,000 110,000 1 Virus yield in plaque-forming units. 264 G r iffith /N o rins/ K agan K aplan el al. [9] performed even more definitive studies showing the change in amino acid utilization when tissue culture cells were infected with herpesvirus. Using ‘tagged' amino acids he found that during the 3- to 4-hour period before cytopathogenicity became apparent, the infected cell machinery, under the influence of the viral DNA, had shifted to making proteins with a preponderance of arginine over lysine. Arginine at the peak of its incorporation into proteins was 125% of the control while lysine utilization dropped to less than 40%. He concluded that his results showing the change in arginine utilization to lysine utilization was a switchover in the manufacture of cell-specific proteins to virus-specific proteins. This verified the shift, or switchover, of codon doublets brought about by the presence of the viral DNA CG content as suggested by Subak-Sharpe et al. [27], The rapid ‘take-over’ of the herpesvirus suggests that arginine might act as an operon coordinate inducer. A preponderance of lysine over arginine could either act as an allosteric enzyme inhibitor [4] or an operon coordinate repressor [15] of the capsid protein synthesis. The studies by T ankersley [28] showing that the lysine must be incor­ porated in the medium before inoculation to prevent viral replication and our clinical studies showing relapse when lysine is discontinued, emphasize that the effect of lysine is only suppressive, not curative. This phenomenon could explain the intermittent characteristic of herpes infection. Table III. Comparison of lysine-arginine in food (g/100 g of food)1 Arginine Lysine Milk 0.12 0.24 Meat 1.2 1.6 Eggs 0.7 0.7 3.7 Brewer’s yeast 2.1 Potatoes 0.1 0.2 Peas 1.6 1.5 Cereal2 0.6 0.4 Peanuts 7.4 2.4 Chocolate 4.0 2.0 1 Adapted from data in reference 6. 2 Nontoasted. A Multiccntcred Study of Lysine Therapy in Herpes simplex Infection 265 The diet of man contains L-lysine and L-arginine. Certain foods are low in lysine but have a relatively high arginine content; others are low in arginine (table III) [6]. Lysine can be synthesized by yeast cells. However, tissues of man apparently cannot synthesize lysine. Once lysine has lost its nitrogen, it cannot be reaminated. Man is totally dependent on his diet as a source of lysine [13]. Variations in absorption and excretion of lysine have been reported [21, 24, 25, 30]. Improper food selection may make adequate lysine intake precarious for some individuals [16]. The ingestion of cereals, seeds, nuts, chocolate, etc., should produce a high arginine to lysine ratio and favor herpetic lesions similar to the results obtained by adding arginine to the medium in the laboratory to induce herpes proliferation. C ontrari­ wise, the avoidance of these foods, coupled with the selection of foods with adequate lysine, such as dairy products, yeast, etc., should discourage herpes infection. This may explain the low incidence of herpes in infants prior to weening from a predominantly milk diet. Our studies indicate that relatively low doses of lysine added to that in the diet can offset the arginine effect. It is interesting that it has been shown since 1952 that lysine has a deterrent effect on viral replication in tissue cultures and since 1964 that arginine has an enhancing effect. Yet there were no clinical studies until K agan [8] reported in 1974 that lysine suppressed herpesvirus infections in patients. For several years we evaluated a herpes simplex vaccine that makes it possible to historically compare the relative merits of lysine therapy. Our results are so encouraging that more elaborate controlled studies, including the pharmacology of lysine, are now underway. References 1 Bud d in g h. C.J.; Schrum. D .I.; L andier. J.C., and G uidry, D.J.: Studies of the natural history of herpes simplex infections. Pediatrics, Springfield //.- 595-610 (1973). 2 B usch, H.: Histones and other nuclear proteins, p. 45 (Academic Press, New York 1965). 3 C h' ien, L.T.; C annon, N .J.; C haramella, L.J.; Dismukes, W.E.; W hitley, R.J.; B uchanan, R.A., and A lford, C.A., j r . : Effect of adenine arabinoside on severe herpesvirus hominis infection in man. J. infect. Dis. 128: 658-663 (1973). 4 Coh en, G .H .: Ribonucleotide reductase activity of synchronized KB cells infected with herpes simplex virus. J. Virol. 9: 408-418 (1972). 5 Editorial: Herpes virus. Latency in neural cells shown in animal models. Hospital Pract. 7: 32 (1972). 266 G r iffith /N o rin s/ K aoan 6 Heinz Nutritional Data; 6th ed. (Heinz International Research Center, Pittsburgh 1972). 7 J aw etz, E.; M elnick, J.L., and A delberg, E.A.: Review of medical microbiology; I Ith cd., pp. 436-437 (Lange Medical Publications, Los Altos 1974). 8 K agan, C. : Lysine therapy for herpes simplex. Lancet i: 137 (1974). 9 K aplan, A.S. ; Shimono, H., and Ben-Porat, T. ; Synthesis of proteins in cells infect­ ed with herpesvirus. III. Relative amino acid content of various proteins formed after infection. Virology 40: 90-101 (1970). 10 K aufman, H.E. : Clinical cure of herpes simplex keratitis by 5-iodo-2'deoxy-uridine. Proc. Soc. exp. Biol. Med. 109: 251 252 (1962). 11 K aufman, H.E.; E llison, E.D., and Tow nsend, W.M.: The chemotherapy of herpes iritis with adenine arabinoside and cytarabine. Archs Ophthal., N.Y. 84: 783-787 (1970). 12 Kinkade, J.M ., jr. : Differences in the quantitative distribution o f lysine-rich histones in neoplastic and normal tissues. Proc. Soc. exp. Biol. Med. 137: 113 1 — 1134 (1971). 13 K leiner, LS. and O rten, J.M .: Biochemistry; 6th ed., p. 484 (Mosby, St. Louis 1962). 14 Lehninger, A.L. : Biochemistry, the molecular basis of cell structure and function, p. 718 (Worth Publishers, New York 1970). 15 L ehninger, A.L. : Biochemistry, the molecular basis o f cell structure and function, p. 734 (Worth Publishers, New York 1970). 16 M ertz, E.T.: Genetic improvement of cereals. Nutr. Rev. 32: 129-131 (1974). 17 M irsky, A.E.; Bur dick, C.J.; D avidson, E.H., and Lillau, V.C.: The role of lysine- rich histones in the maintenance of chromatin structure in metaphase chromosomes. Proc. natn. Acad. Sci. USA 61: 592 597 (1968). 18 M urray, K.: The heterogenicity of histones; in Bonnf.r and Ts'o The nucleohistones, p. 24 (Holden-Day, San Francisco 1964). 19 M yers, M .G.; D aman, N.M .; C lark, J.E., and A rndt, K.A.: Failure of neutral red photodynamic inactivation in recurrent herpes simplex virus infections. New Engl. J. Med. 293: 945-949 (1975). 20 N ugent, G.R. and C hou, S.M.: Treatment of labial herpes. J. Am. med. Ass. 224: 132 (1973). 21 O mura, K.: Lysine malabsorption syndrome. A new type of transport defect. Pedia­ trics, Springfield 57: 102-105 (1976). 22 Pearson, H.E. ; L agerborg, D.L., and W inzler, R.J.: Effect of certain amino acids and related compounds on propagation of mouse encephalomyelitis virus. Proc. Soc. exp. Biol. Med. 70: 409-411 (1952). 23 Sabin, A.B. : Misery o f recurrent herpes. What to do? New Engl. J. Med. 293: 986-988 (1975). 24 Sh ih, V.E.; G lick, T.H., and Bercu, B.B.: Lysine metabolism in Reye's syndrome. Lancet ii: 163-164 (1974). 25 Simell, O.; Perheentupa, J.; R apola, J.; Visakorpi, J.K., and Eskelin, L.-E.: Lysinuric protein intolerance. Am. J. Med. 59: 229-240 (1975). 26 Subak-S harpe, H. and H ay, J.: An animal virus with DNA of high guanine + cyto­ sine content which codes for s-RNA. J. molec. Biol. !2: 924-928 (1965). 27 S ubak-S harpe, H .; Burk, R.R.; C rawford, L.V.; Morrison, J.M ., an d H ay, J.: An ap p ro ach to evolutionary relationships o f m am m alian DNA viruses through A Multicentered Study of Lysine Therapy in Herpes simplex Infection 267 analyses of the pattern of nearest neighbor base sequences. Quant. Biol. 31: 737-748 (1966). 28 T ankkrsley, R.W., j r . : Amino acid requirements of herpes simplex virus in human cells. J. Bact. 87: 609-613 (1964). 29 T r o pin, J.A.; R ottman, F.M .; Brimacombe, R.L.C.; L eder, P.; Bernfield, M.R., and N irenberg, M.W.: RNA codewords and protein synthesis. VI. On the nucleotide sequences of degenerate codeword sets for isolcucine, tyrosine, asparagine, and lysine. Proc. natn. Acad. Sci. 58: 807-811 (1965). 30 Van G elderen, H.H. and T eijema, H .L .: Hyperlysinaemia. Harmless inborn error of metabolism? Archs Dis. Childh. 48: 892-895 (1973). 31 W agner, R .R .: Herpes simplex: in Beeson and M c D ermott Textbook of medicine, pp. 117-119 (Saunders, Philadelphia 1967). R.S. G riffith, MD, Lilly Research Laboratories, PO Box6 18, Indianapolis, I N 46206(USA)

Journal

Dermatology – Karger

Published: Jan 1, 1978

Keywords: Herpes simplex; Lysine therapy

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