Acr87.tmp

success in complex human diseases such as human im-munodeficiency virus.2 To determine whether such an approach might be effective in ALS, we evaluated thecombination of two agents currently available for hu- man use. Experimental administration of either mino- cycline or creatine has been demonstrated to delay dis-ease onset and extend survival in transgenic mouse Wenhua Zhang, MD, PhD, Malini Narayanan, MS, MD, models of ALS.3–6 Given that these two compounds act by different mechanisms of action, we evaluatedwhether the combination of minocycline with creatinemight result in additive neuroprotective effects in ALS The known neuroprotective effects of minocycline and
mice. We report that the combination of minocycline creatine in animal models of amyotrophic lateral sclerosis
and creatine results in an additive effect, delaying dis- (ALS) led us to examine whether the combination of
ease onset, slowing progression, and delaying mortality.
these agents would result in increased neuroprotection.
Because these two compounds are currently available As previously reported, we confirmed in ALS mice that
for human use, their combined use may be evaluated either minocycline or creatine treatment results in im-
provement in motor performance and extended survival.
We report that combination of minocycline and creatine
resulted in additive neuroprotection, suggesting this to be
Materials and Methods
a novel potential strategy for the treatment of ALS. To
our knowledge, this is the first report demonstrating ad-
Mice carrying the human SOD1G93A mutant gene were ob- ditive neuroprotection of a combinatorial approach in a
tained from Jackson Laboratories (Bar Harbor, ME). Mice mouse model of ALS. Adding relevancy to our findings,
were crossbred and genotyped as previously described.7 Mice minocycline and creatine, are relatively safe, cross the
were fed a diet supplemented with 2% creatine beginning at blood–brain barrier, and are currently available for hu-
3 weeks of age (courtesy of Dr R. Kaddurah-Daouk). Mino- man evaluation.
cycline (22mg/kg body weight/day) was injected intraperito- neally beginning at 4 weeks of age (Sigma, St. Louis, MO).
Minocycline was prepared fresh in 0.9% saline daily. Themonotherapy and untreated groups were fed control diet or Amyotrophic lateral sclerosis (ALS) is a chronic neuro- injected with saline as appropriate in the same schedule asthe treated groups. Experiments were conducted in accor- degenerative disease characterized by progressive motor dance with protocols approved by the Harvard Medical weakness resulting from selective motor neuron cell death.1 Mortality is seen on the average 4 years after The selection of 10 mice per group was determined from disease onset. Proven therapeutic options are limited to our previous experience evaluating minocycline in ALS riluzole, which extends survival by an average of 3 mice.3 In that study, using 10 mice per group, we detected months.1 It is therefore of utmost importance to de- 10% protection mediated by minocycline. Given the above- velop novel and more effective therapeutics for this mentioned information, as well as our hypothesis that the universally fatal devastating disease. Because of the combinatorial regimen would result in improved protection, multifactorial downstream pathogenic pathways acti- we decided to evaluate a group size of 10.
vated in ALS, improving therapeutic efficacy will de-pend on implementing a strategy addressing multiple components of the pathogenesis. Combinatorial thera- Motor strength and coordination were evaluated with a Ro- peutics have been implemented with varying degrees of tarod (Columbus Instruments, Columbus, OH), beginningat 10 weeks of age. Mice were evaluated at 5 and 15rpm.
The time each mouse remained on the rod was registeredautomatically. If the mouse remained on the rod for 7 min-utes, the test was completed and scored as 7 minutes. Mice From the Neuroapoptosis Laboratory, Department of Neurosurgery, were tested weekly until they could no longer perform the Brigham and Women’s Hospital, Harvard Medical School, Boston, task. Two investigators performed the experiment, one eval- uated Rotarod performance and was blinded to mice treat- Received Jul 17, 2002, and in revised form Nov 5. Accepted for ments (M.N.), and the other treated the mice (W.Z.).
Onset and SurvivalDisease onset was defined as the first day that a mouse could Address correspondence to Dr Friedlander, Neuroapoptosis Labora- not remain on the Rotarod for 7 minutes at 15rpm. Mor- tory, Department of Neurosurgery, Brigham and Women’s Hospi- tality was scored as the age of death or the age when the tal, Harvard Medical School, Boston, MA 02115.
E-mail: rfriedlander@rics.bwh.harvard.edu mouse was unable to right itself within 30 seconds.
Statistical AnalysisThe findings were reported as the mean Ϯ standard devia-tion. Statistical comparisons between control group andtreated groups were performed using one-way analysis ofvariance.
Results
As previously reported, mutant SOD1G93A mice treated
with either intraperitoneal injections of minocycline or
2% oral creatine demonstrated a significant delay of
disease onset and increased survival as compared with
mice fed unsupplemented diets and injected with saline
(Table).3,4,5,6 Disease onset was delayed to 113 and
111 days, respectively, in the minocycline- and
creatine-treated groups as compared with 94 days in
the control group ( p Ͻ 0.05). Survival was similarly
delayed in the minocycline and creatine groups to 142
Fig. Cumulative probability of onset of Rotarod deficits (a) and 141 days, respectively, as compared with 126 days and survival (b) in amyotrophic lateral sclerosis (ALS) mice. in the control group ( p Ͻ 0.05). Minocycline-creatine Survival was significantly prolonged and the onset of Rotarod combination resulted in additive neuroprotection, with deficit was significantly delayed in ALS mice treated with mi- disease onset detected at 122 days and mortality at 157 nocycline and/or creatine when compared with saline-treatedtransgenic littermates. Combination of minocycline with creat- days. The differences of onset and survival between the ine significantly delayed onset and mortality as compared with monotherapy groups and the minocycline-creatine minocycline or creatine alone. Motor function was tested with group were significantly different ( p Ͻ 0.05). Survival the Rotarod at 5 (c) and 15rpm (d). Testing was terminated was extended by 13%, 12%, and 25% in the minocy- either when the mouse fell from the rod or at 7 minutes if the cline, creatine, and minocycline-creatine groups, re- mouse remained on the rod. Mice treated with the spectively, as compared with the control group.
minocycline-creatine combination performed significantly better Kaplan–Meier curves of disease onset and survival as than mice treated with either compound alone ([pound sign] well as plots of Rotarod performance clearly demon- p Ͻ 0.05). Mice treated with either minocycline or creatine strate the additive protection of the minocycline- performed significantly better than control mice ([asterisks] p creatine combination (Fig). At the doses and schedule Ͻ 0.05, n ϭ 10 per group). tested, there were no significant differences in motorperformance or disease progression between the mino- these abnormalities are interrelated, although a direct cause and effect relationship cannot always be clearlydelineated. Therapeutic approaches targeting only one Discussion
of the above-described abnormalities is likely to have Successful combinatorial approaches rely on the use of only a very limited impact on the progression of the agents with different but complementary mechanisms disease. Similar to what has been successfully executed of action. This approach is of particular importance in cancer and human immunodeficiency virus, a com- when treating diseases with complex and multiple bination of drugs targeting different components of the pathologic abnormalities. A broad variety of cellular particular pathophysiology of the disease will result in abnormalities have been described in human specimens enhanced effects as compared with monotherapeutic and mouse models of ALS. Broadly, documented ab- approaches.2 Using the incremental knowledge we have normalities in ALS include apoptotic, bioenergetic, ex- acquired regarding the mechanisms of pathogenesis in citotoxic, and mitochondrial pathways.1,3,4,7–9 Many of ALS, we are in the position to design rational thera- Table. Onset of Motor Deficits and Mortality of ALS Mice Treated with Minocycline and/or Creatine (mean Ϯ SD) ap Ͻ 0.05 compared with saline group.
bp Ͻ 0.05 compared with minocycline and with creatine group.
ALS ϭ amyotrophic lateral sclerosis; SD ϭ standard deviation.
peutics, targeting different components of this disease.
studies have been done in single therapy of creatine4 The result of these experiments is the first demonstra- and minocycline5 in mSOD1G93A transgenic mice.
tion of the efficacy of this approach in ALS. Further- Klivenyi and colleagues demonstrated a remarkable in- more, many patients with ALS are likely taking com- hibition of motor neuron cell loss in creatine-treated binations of medications, with no objective evidence of ALS mice as compared with control ALS littermate any benefit, and exposing themselves to potential for mice.4 Furthermore, Van den Bosch and colleagues re- harmful interactions or additive side effects.
ported decreased microglial activation in spinal cord.5 Minocycline is a second-generation tetracycline, cur- Synergistic protection of motor neurons could likely rently used in the treatment of certain infections, acne, explain the additive neuroprotection detected in this and rheumatoid arthritis. Minocycline is used as a study using minocycline and creatine.
chronic medication in the latter two diseases. Cur- For the first time to our knowledge, we report effec- rently, the mode of action of minocycline appears to be tive additive neuroprotection in a therapeutic trial us- multifactorial. We recently have demonstrated that a ing ALS mice. Note that the ability to rationally design direct action of minocycline is inhibition of mitochon- such studies has depended on incremental understand- drial permeability transition–mediated release of cyto- ing of the mechanisms of disease progression in ALS, chrome c.3 Given that release of cytochrome c is a crit- as well as the mechanisms of action of minocycline and ical component of the apoptotic cascade, inhibition creatine. Even greater neuroprotection than that dem- of cytochrome c release results in inhibition of cell onstrated from the minocycline-creatine combination death. Additional activities have been associated with might result from including additional compounds minocycline-mediated neuroprotection including inhi- with complementary mechanisms of action. In light of bition of reactive microgliosis, of caspase-1, caspase-3, the above described results, the relative safety record of and nitric oxide synthase transcriptional upregulation minocycline and creatine, and their ability to cross the and of p38 MAPK activation.9–13 Whether any of blood–brain barrier, their combined use may be evalu- these properties of minocycline are direct or reactive in nature remains to be elucidated. In addition to ALS,minocycline thus far has been demonstrated to be ef-fective in experimental models of stroke, Huntington’s This work was supported by grants from the NINDS (NS41635 disease, Parkinson’s disease, and trauma.10–12,14 Creatine is an amino acid endogenously produced from glycine, methionine, and arginine in the liver,kidney, and pancreas. It has remarkable neuroprotec- References
tive qualities in models of ALS, traumatic brain injury, 1. Rowland LP, Shneider NA. Amyotrophic lateral sclerosis.
Huntington’s disease, and Parkinson’s disease.4,15–17 2. Temesgen Z, Wright AJ. Antiretrovirals. Mayo Clin Proc 1999; Currently, the mechanism of creatine-mediated neuro- protection is not well understood. Creatine kinase 3. Zhu S, Stavrovskaya IG, Drozda M, et al. Minocycline inhibits along with its substrates creatine and phosphocreatine cytochrome c release and delays progression of amyotrophic lat- constitute key components of cellular bioenergetic eral sclerosis in mice. Nature 2002;417:74 –78.
pathways.4 The mechanistic basis for the neuroprotec- 4. Klivenyi P, Ferrante RJ, Matthews RT, et al. Neuroprotective effects of creatine in a transgenic animal model of amyotrophic tive effects of creatine may involve alterations of the lateral sclerosis. Nat Med 1999;5:347–350.
insult-induced depletion of cellular ATP, because 5. Van Den Bosch L, Tilkin P, Lemmens G, Robberecht W. Mi- chronic ingestion of creatine results in increased brain nocycline delays disease onset and mortality in a transgenic levels of phosphocreatine. To this regard, creatine pro- model of ALS. Neuroreport 2002;13:1067–1070.
tects mitochondrial creatine kinase activity against 6. Kriz J NM, Nguyen M, Julien J. Minocycline slows disease pro- gression in a mouse model of amytrophic lateral sclerosis. Neu- peroxynitrite-induced inactivation, which may play a role in tissue damage in ALS.18 Most recently, direct 7. Gurney ME, Pu H, Chiu AY, et al. Motor neuron degeneration antioxidant properties of creatine have been described in mice that express a human Cu,Zn superoxide dismutase mu- that may be relevant to explain some of its neuropro- tation. Science 1994;264:1772–1775.
tective effects.19 Creatine has been evaluated in human 8. Friedlander RM, Brown RH, Gagliardini V, et al. Inhibition of ICE slows ALS in mice. Nature 1997;388:31.
ALS trials. Preliminary results demonstrated that crea- 9. Martin LJ. Neuronal death in amyotrophic lateral sclerosis is tine supplementation temporarily increases maximal apoptosis: possible contribution of a programmed cell death mechanism. J Neuropathol Exp Neurol 1999;58:459 – 471.
This study does not address neuropathological 10. Yrjanheikki J, Keinanen R, Pellikka M, et al. Tetracyclines in- changes in combined therapy compared with mono- hibit microglial activation and are neuroprotective in global brainischemia. Proc Natl Acad Sci USA 1998;95:15769 –15774.
therapy. However, neuropathological studies may help 11. Chen M, Ona VO, Li M, et al. Minocycline inhibits caspase-1 elucidate the mechanism of additive neuroprotection and caspase-3 expression and delays mortality in a transgenic using minocycline and creatine. Neuropathological mouse model of Huntington disease. Nat Med 2000;6:797– 801.
12. Du Y, Ma Z, Lin S, et al. Minocycline prevents nigrostriatal do- phagia in 8 patients, also were observed among our
paminergic neurodegeneration in the MPTP model of Parkin- LEMS population. More than one prereferral oculobul-
son’s disease. Proc Natl Acad Sci USA 2001;98:14669 –14674.
bar feature occurred in 13 of our LEMS patients. Prere-
13. Almer G, Vukosavic S, Romero N, Przedborski S. Inducible nitric oxide synthase up-regulation in a transgenic mouse model ferral diagnostic considerations included myasthenia gra-
of familial amyotrophic lateral sclerosis. J Neurochem 1999;72: vis, myopathies, and psychiatric disorders. These findings
suggest that these atypical characteristics served to dis-
14. Sanchez Mejia RO, Ona VO, Li M, Friedlander RM. Minocy- suade some colleagues from a diagnosis of LEMS. Thus,
cline reduces traumatic brain injury-mediated caspase-1 activa- the presence of oculobulbar symptoms and signs cannot
tion, tissue damage, and neurological dysfunction. Neurosur- be used to exclude LEMS from the differential diagnosis.
gery 2001;48:1393–1399; discussion, 1399 –1401.
15. Sullivan PG, Geiger JD, Mattson MP, Scheff SW. Dietary sup- plement creatine protects against traumatic brain injury. AnnNeurol 2000;48:723–729.
16. Ferrante RJ, Andreassen OA, Jenkins BG, et al. Neuroprotec- tive effects of creatine in a transgenic mouse model of Hunting- The Lambert–Eaton myasthenic syndrome (LEMS) is ton’s disease. J Neurosci 2000;20:4389 – 4397.
an autoimmune disorder of neuromuscular transmis- 17. Matthews RT, Ferrante RJ, Klivenyi P, et al. Creatine and cy- sion.1–3 Typically when LEMS is related to a malig- clocreatine attenuate MPTP neurotoxicity. Exp Neurol 1999;157:142–149.
nancy, it is almost always a small cell lung cancer 18. Wendt S, Dedeoglu A, Speer O, et al. Reduced creatine kinase (SCLC). Although LEMS is sometimes associated with activity in transgenic amyotrophic lateral sclerosis mice. Free other neoplasms, 15% of patients with other SCLC- 19. Lawler JM, Barnes WS, Wu G, et al. Direct antioxidant prop- related autoimmune disorders have a coexisting, unre- erties of creatine. Biochem Biophys Res Commun 2002;290: lated, and often obvious neoplasm.4–13 When there is no paraneoplastic mechanism identified, LEMS usually 20. Mazzini L, Balzarini C, Colombo R, et al. Effects of creatine has a primary autoimmune basis sometimes associated supplementation on exercise performance and muscularstrength in amyotrophic lateral sclerosis: preliminary results.
with other immunological disorders.7,10 The neuro- physiological characteristics of LEMS represent theclinical prototype of a presynaptic neuromusculartransmission disorder.1,4-6 The voltage-gated calciumchannel of motor nerve terminals is the primary site ofimmune attack.3,8,9 Classically, LEMS presents with fatigability, proxi- mal weakness, hyporeflexia or areflexia of the muscle stretch reflexes, and cholinergic dysautonomia.4–7,14Oculobulbar symptoms are reported to be less com- mon and mild and therefore have not received much Ted M. Burns, MD,1 James A. Russell, MD,1 attention.6,7,14 The diagnosis of LEMS is sometimes quite challenging. This is because of its relative rarity, the nonspecificity of symptoms with fatigue as a pri-mary clinical complaint, often the generally mild na-ture of objective weakness, and because the symptoms Oculobulbar symptoms and/or signs were present in 18
of dysautonomia are often overlooked. Last, the pro- of 23 (78%) of Lambert–Eaton myasthenic syndrome
(LEMS) patients evaluated at the Lahey Clinic (Table).
tean clinical presentations of LEMS are not always ap- Sixty-five percent (15 of 23) of our patients had ptosis
preciated. Because of the therapeutic importance inher- and/or diplopia, each present in 11 individuals. Bulbar
ent in the early recognition of LEMS, it is incumbent signs and symptoms, including dysarthria in 10 and dys-
on the neurologist to be aware of these variable clinicalpresentations. Because it is our clinical experience thatLEMS may be underrecognized and its diagnosis is of-ten delayed, we have reviewed our series of 23 LEMS From the 1Department of Neurology, Lahey Clinic, Burlington,MA, and the 2Department of Neurology, Mayo Clinic, Rochester, patients diagnosed at the Lahey Clinic. These data demonstrate that oculobulbar involvement, so typical Received Jun 14, 2002, and in revised form Nov 11. Accepted for of myasthenia gravis (MG), is also a more common clinical finding of LEMS than is emphasized in theclassic teaching of this entity. Recognition of this ob-servation may enhance an earlier diagnosis of the Address correspondence to Dr Burns, Lahey Clinic, 41 Mall Road,Burlington, MA 01805. E-mail: tmb8r@virginia.edu

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