Efficacy of rintatolimod in the treatment of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME)

Figures & data

Figure 1. MyD88 dependent and Myd88 independent signaling pathways for the TLRs and helicases. A. Intracellular pathways for MyD88 independent TLR3 nuclear signal transduction initiated by TRIF binding to the TIR of the TLR3 homodimer. TLR3 monomers dimerize with binding of the dsRNA ligand. Activated TRIF initiates two pathways. The first results in the transitory induction of the IFNs. The second is a species variable pathway (rodents ≫primates) that operates though NFκB (dashed line), which transiently induces the production of inflammatory cytokines. The adapter protein cascade initiated by TRIF (TIR-domain-containing adapter-inducing interferon) includes TBK1 (TANK-binding kinase 1 binds to TRAF3), TRAF1/3 (TNF receptor associated factors), NAP1 (Nck-associated protein 1), IKK (IκB kinase), IKKε (inhibitor of IκB kinase), P13K (Phosphoinositide 3-kinase), IRF3/7 (interferon regulatory transcription factors), TAK1 (protein kinase of MLK family), TAB1 (TGF-β activated kinase 1), RIP1 (Receptor-interacting [TNFRSF] kinase 1), NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells), IκB (inhibitor NFkB). The ectodomain of TLR3 consists of a horseshoe shaped structure populated by 23 leucine-rich β-sheets (orange disks) connected by non-ordered chains containing RNA binding residues. The transmembrane a-helices (solid orange) connect the ectodomain to the cytoplasmic TIR domain (dark green). The phosphorylated TIR binds TRIF to initiate the adapter protein cascade. B. Intracellular pathways for MyD88 dependent for TLR 1/2 and 1/6 heterodimers and TLR 4–10 homodimers with the diverse PAMP ligands represented by a green bar is not necessarily as accurate in placement as is dsRNA with TLR3 in 1A. TLR4 uses both the MyD88 dependent and independent pathways. Reproduced from Mitchell WM, et al .Discordant Biological and Toxicological Species Responses to TLR3 Activation. Am J Path 2014; 184: 1062–72.

Table 1. Characteristics and specifications for clinical grade rintatolimod.

Generic name: Rintatolimod

Brand name: Ampligen

Molecular Formula

Poly I: Poly C12U is designated by the molecular formula, [rI(13):rC(12)rU(1)]n where: n = 46–138, or (C10 H11 N4 O7 P)13: (C9 H12 N3 O7 P)12 (C9 H11 N2 O8 P).

Molecular Weight

400,000–1,200,000 (10.0–15.0 S20w)

Chemical Abstracts (CAS) Registry Number

38640–92-5

Chemical Name

• Chemical abstracts names: 5ʹ-Inosinic acid, homopolymer, complex with 5ʹ-cytidylic acid polymer with 5ʹ-uridylic acid (1:1) (9CI) (CA INDEX NAME) 5ʹ-Cytidylic acid, polymer with 5ʹ-uridylic acid, complex with 5ʹ-inosinic acid homopolymer (1:1) (9CI) 5ʹ-Uridylic acid, polymer with 5ʹ-cytidylic acid, complex with 5ʹ-inosinic acid homopolymer (1:1) (9CI) Ampligen Poly(I).poly(C12U)

• Other names: Polyriboinosinic-polyribocytidylic 12: uridylic acid; Poly I: Poly C12U Rintatolimod

Nucleotide composition

The ratio of poly I to poly C12U is 0.9–1.1–1.0

Formulation

Rintatolimod is supplied in glass bottles as a biological active (TLR3 agonist in Ramos-Blue^TM reporter cells), sterile, colorless solution containing 200 mg of Poly I: Poly C12U in 80 ml of a physiological solution of salts (0.15 M NaCl, 0.01 M Phosphate, 0.001 Mg^++) at a concentration of 2.5 mg/ml (2.25–2.70 mg/ml) with tertiary conformation demonstrated by circular dichroism. The product does not contain preservatives or antioxidants and is free of particulates (Particles ≥10 µm < 3000 per container; Particles ≥25 µm < 300 per container) and endotoxins (LAL activity < 1 EU/mL). The shelf life at 2–8°C is >7 years.

Figure 2. Diagramatic representation of rintatolimod. The dsRNA structure is maintained by hydrogen bonding. The introduction of a uracil into the poly C strand provides thermodynamic instability with an increased susceptibility to blood nuclease hydrolysis. The poly I strand is represented by blue (inosine). The poly C₁₂U bases are represented by green (cytosine) and red (uracil).

Figure 3. Representative rintatolimod blood elimination curves: (a) 200 mg (n = 6), (b) 400 mg (n = 12), and (c) 700 mg (n = 4) dose groups. Rintatolimod was delivered as a single bolus over an infusion period of 23–60 minutes. Rintatolimod and its metabolites (>50–100 nucleotides) were determined by hybridization employing a [³H]poly(C) probe. Each point is the average of duplicate concentration determinations at the designated dose and within 2.5 minutes of the time indicated. Each curve is the product of ‘n’ infusions, using first order decay kinetics. Revised from the doctoral thesis of Kenneth Strauss [25] with permission from the author.

Figure 4. Molecular Model of the human TLR3 dimer ecodomain and its rintatolimod ligand. Figure 4(a) is viewed from a lateral view of rintatolimod bound to the active site of the TLR3 homodimer. The C-terminal regions of each dimer face each other and bind to the phosphate backbone of the dsRNA. The N-terminals of each TLR3 bind to opposite ends of the dsRNA with a minimum length of 45 bp required for interaction with essential residues of TLR3 for activation of intracellular signaling. Amino acids of TLR3 required for binding of rintatolimod are shown as CPK (Van der Waals’ radii) associated with the phosphate backbone. Figure 4(b) illustrates the TLR3 homodimer complexed with rintatolimod as seen down the long axis of the dsRNA. The TLR3 homodimers are represented as structural elements with the blue arrows signifying direction of β-sheets and the red cylinders signifying α-helices. The Poly I strand of rintatolimod is colored blue and the poly C12U strand magenta. Reproduced from Mitchell WM, et al. Discordant Biological and Toxicological Species Responses to TLR3 Activation. Am J Path 2014; 184: 1062–72.

Table 2. Rintatolimod open-label studies.

Protocol	No. of patients treated	Percent female	Observed safety and apparent efficacy
AMP 501 Phase I	14	75	Drug was well tolerated throughout study (majority >24 weeks) in a relatively homogenous CFS/ME cohort. Efficacy observed in exercise performance, neurocognition, and anti-HHV-6 activity [41].
AMP 502E Phase I/ II	22	73	Open-label extension study in a 22 patient cohort who completed double-blind AMP 502 treated during an open-label extension phase for 1 year or longer. Drug was well tolerated throughout study (Phase 1 extention of administration time. Extention (Phase II) provided major improvements in Karnoksky Performance Status (p < 0.0001) [43].
AMP 509 Phase II	45	70	Belgian open-label study, with 44 patients evaluated. Similar dosing procedure and endpoints as AMP 502 [42]
AMP 511 Phase II/III	139	65	Ongoing cost-recovery, open-label study of Safety and Efficacy with similar dosing procedures and endpoints as AMP 502 [42]
AMP 516E Phase III	190	73	Partially blinded cross-over cohort extension of double-blind AMP 516 study [44]
Total	412	70

Table 3. Rintatolimod placebo-controlled studies [43,44].

Protocol	No. of patients	Percent female	Study design/data reported
AMP 502 Phase II	92	75	Placebo-controlled, randomized, multicenter Safety and Efficacy Evaluated Placebo (n = 47) or rintatolimod (n = 45): 200 mg for their first four doses, then 400 mg twice a week for 6 months
AMP 502 T Phase II	19	74	Placebo-controlled, randomized, multicenter Safety Evaluated 10 patients received placebo and 9 patients were treated with a higher dosing schedule of rintatolimod (400 mg three times a week)
AMP 516 Phase III	234	73	Placebo-controlled, randomized, multicenter, Safety and Efficacy Evaluated Placebo (n = 117) or rintatolimod (n = 117) 200 mg twice weekly for first four doses, then 400 mg twice weekly for 40 weeks (Stage I)
Total	345	74

Table 4. AMP-502 clinical trial differential responses^a.

	Percentage change (n)
Parameter	Rintatolimod	Placebo	p-Value
KPS	+20 (41)	0 (43)	<0.001 (mean)/0.023 (median)^b
Cognitive deficit	+27.3 (40)	+14.5 (43)	0.05^c
ADL status	+23.1 (41)	+ 14.1 (43)	0.034^c
Exercise duration	+10.3 (37)	+ 2.1 (39)	0.007^d
Exercise work	+11.8 (37)	+5.8 (39)	0.011^e

^aModified from [24] (Clinical Infectious Diseases/Oxford University Press).

^bMedian change (Week 24 vs. baseline) by Mann–Whitney test.

^cAn increased score quantifies a reduction in perceived deficit.

^dANCOVA with baseline as covariate.

^eANCOVA of log-transformed data with baseline as covariate.

Table 5. AMP-502 differential medication usages.

	Percentage change (n)
Drug class	Rintatolimod	Placebo	p-Value^a
CFS/ME symptoms	0.05	1.0	0.015
CNS symptoms	0.03	0.43	0.033
Pain	0.04	1.1	0.009
All medications	0.44	2.3	0.007

^aMean use during the first 4 weeks vs. last 4 weeks (t-test).

Table 6. AMP-502-relative incidence of hospitalizations^a in CFS/ME patients receiving rintatolimod vs. placebo.

		Rintatolimod	Placebo	Mann–Whitney
Number of admissions per hospitalized/ER patient	Mean	1.0	3.4	p < 0.005
	Median	1.0	3.0
Number of days per hospitalized/ER patient	Mean	2.7	16.3	p < 0.005
	Median	1.0	18.0

^aHospitalization was defined as either an emergency room admission or an admission to the inpatient service.

Table 7. AMP-516 analysis of the effect of rintatolimod on the primary endpoint, Exercise Tolerance (ET) [43,44].

A. Increase in exercise treadmill duration with rintatolimod in CFS patients (intent-to-treat)
	Mean (SD) exercise duration (Seconds)		Percent increase from baseline^a
Study interval	Rintatolimod(n = 100)	Placebo (n = 108)	Rintatolimod (n = 100)	Placebo (n = 108)	p-value
Baseline	576 (257.5)	588 (234.4)	-	-	0.729^b
Week 40	672 (314.1)	616 (286.7)	36.5	15.2	0.047^c
p-value^d			<0.001	0.198
B. Increase in exercise treadmill duration with rintatolimod in CFS patients (trial completion population)
	Mean (SD) exercise duration (Seconds)		Percent increase from Baseline^a		p-value
Study interval	Rintatolimod (n = 93)	Placebo (n = 101)	Rintatolimod (n = 93)	Placebo (n = 101)
Baseline	583 (254.7)	587 (237.3)	-	-	0.908^b
Week 40	691 (311.4)	614 (291.2)	40.2	15.6	0.019^c
p-value^d			<0.001	0.244
C. Increase in exercise treadmill duration with rintatolimod in CFS patients without significant dose reductions (intent-to-treat)
	Mean (SD) exercise duration (Seconds)		Percent increase from baseline^a		p-value
Study interval	Rintatolimod (n = 83)	Placebo (n = 98)	Rintatolimod (n = 83)	Placebo (n = 98)
Baseline	581 (256.2)	590 (235.3)	-	-	0.813^b
Week 40	690 (308.2)	616 (291.4)	43.0	15.0	0.022^c
p-value^d			<0.001	0.263
D. Effect of baseline ET on Week 40 ET (intent-to-treat)
	Mean (SD) exercise duration mean (seconds)				% Gain Rintatolimod over Placebo^a		p-value^c
Baseline ET Strata (Minutes)	≤ 9 Drug (n = 40)	≤ 9 Placebo (n = 42)	> 9 Drug (n = 60)	> 9 Placebo (n = 66)	≤ 9	> 9	≤ 9	> 9
Baseline	321 (153.3)	353 (144.6)	747 (148.3)	738 (137.7)
Week 40	450 (284.2)	446 (264.6)	820 (237.3)	725 (245.9)	31.0	15.0	0.517	0.034
E. Mean/Median baseline and Mean/Median change from baseline in exercise treadmill duration (seconds) at Week 24 (Stage 2) (ITT population)
	Rintatolimod to Rintatolimod		Placebo to Rintatolimod
Exercise tolerance parameter	Mean (SD)	Median	Mean (SD)	Median
Baseline ET (seconds)	706 (308)	726	626 (291)	638
ET at 24 weeks (seconds)	696 (323)	732	669 (288)	665
Percent improvement	22.9	-	39.0	-
p-value^c	0.58	0.69	0.04	0.02

^aMean intra-patient percent improvement. ^bStudent’s t-test comparing mean baseline ET between treatment groups. ^cAnalysis of covariance (ANCOVA) with baseline as a covariate comparing the mean ET change from baseline within each treatment group. ^dPaired t-test comparing whether the change from baseline is equal to zero within each treatment group.

Figure 5. AMP-516 plot of ET difference from baseline in seconds at 40 weeks treatment (ordinate) per each patient (abscissa). Plot of ET difference from baseline in seconds at 40 weeks treatment (ordinate) per each patient (abscissa). Reproduced from Strayer et al. Chronic Fatigue Syndrome/ Myalgic Encephalomyelitis (CFS/ME): Characteristics of responders to Rintatolimod. J Drug Res Dev 2015;1: doi http://dx.doiorg/10.16966/jdrd.103.

Table 8. Analysis of percentage of CFS/ME patients improving ET by at least 25% and 50% from baseline [43].

	% of Patients (n) improving
Percent improvement	Rintatolimod	Placebo	p-value^a
A. Intention to treat (ITT) population (n = 208)
≥25%	39% (n = 39)	23.1% (n = 25)	0.013
≥50%	26% (n = 26)	13.9% (n = 15)	0.028
B. Subsets of ITT population with baseline ET>9 minutes (n = 126)
≥25%	33.3% (n = 20)	12.1% (n = 8)	0.004
≥50%	23.3% (n = 14)	4.5% (n = 3)	0.003

^aProbability values derived from the Chi-square test or Fisher’s Exact Test if any cell had less than 5 observations.

Table 9. AMP-516 maximal oxygen utilization (VO₂ max) in >9 minute baseline cohort.

	VO2 max mean (ml/min/kg)		Percent increase from baseline
Week	Rintatolimod	Placebo	Rintatolimod	Placebo	p-value^a
Baseline	22.39	21.70	__	__	__
	(n = 58)	(n = 60)
Week 40	22.75	20.86	1.61	−3.87	0.05
	(n = 58)	(n = 62)

^aAnalysis of Covariance (Baseline as Covariate), log transformed valves; High Stratum (Baseline > 9 minutes).

Table 10. Summary of all patients with adverse events with at least 5% difference between rintatolimod and placebo.

Patients with any adverse events		Controlled portions of Phase II/III clinical trials
		Studies AMP-502 and AMP-516
		Rintatolimod	Placebo
		(N = 162)	(N = 164)
		161 (99.4 %)	160 (97.6 %)
Adverse events	% Difference*
Flu-like symptoms	13.9	72 (44.4 %)	50 (30.5 %)
Headache	12.8	74 (45.7 %)	54 (32.9 %)
Chills	9.4	27 (16.7 %)	12 (7.3 %)
Fever	8.2	33 (20.4 %)	20 (12.2 %)
Vasodilatation	7.6	27 (16.7 %)	15 (9.1 %)
Pain	7.3	75 (46.3 %)	64 (39.0 %)
Injection site reaction	7.1	50 (30.9 %)	39 (23.8 %)
Pruritus	7.0	33 (20.4 %)	22 (13.4 %)
Diarrhea	6.3	36 (22.2 %)	26 (15.9 %)
Syncope	6.2	13 (8.0 %)	3 (1.8 %)
Ear disorder	5.7	22 (13.6 %)	13 (7.9 %)
Nausea	5.4	67 (41.4 %)	59 (36.0 %)
Migraine	−5.3	16 (9.9 %)	25 (15.2 %)

Table 11. Relationship of SAEs to study drug as determined by blinded investigators.

	Number of serious adverse events^a
Relationship to study drug	Rintatolimod	Placebo
Not related	16	16
Remote	4	3
Possible	2	2
Probable	0	1
Definite	0	0
Total	22	22

^aDetermined at time of occurrence.

Table 12. Comparative species sensitivity to rintatolimod^a. [20].

Species	Maximum tolerated dose
Rabbit	1.25 mg/kg/dose
Dog	10 mg/kg/dose
Rat	12.5 mg/kg
Cynomolgus monkey	100 mg/k g

^aThe MTD is defined as the highest dose with no observed mortality or moribund toxicity.

Table 13. Rintatolimod mutagenic potential.

Mutagenicity assay	Result
Mammalian Cytogenetic Assay (CHO Cells)	Negative
Ames Assay	Negative
L5178Y TK ± Mouse Lymphoma Mutagenesis	Negative
Mouse Bone Marrow Micronucleus Assay	Negative

Table 14. Evidence-based potential diagnostic markers for rintatolimod response in CFS/ME.

Marker	Rationale	Improvements
ET>9 minutes on modified Bruce protocol	Demonstrated in Phase II and Phase III clinical trials	ET under measurement of O2 utilization and CO2 production to insure acquisition of ‘oxygen debt’ as objective evidence of termination due to exhaustion
Low NK cell function enhancement	Low NK cell functional activity demonstrated in multiple studies. Low NK cell activity up-regulated with rintatolimod in vitro	In vitro response has not been correlated with in vivo response in CFS/ME patients. Cr^51 release assay from NK cell target cells in vitro is a research lab assay. NK cell markers used in flow cytometry with fixed cells needs correlation with Cr^51 release response to rintatolimod
37kD RNAse L	Low MW proteolytic form of RNAse L in CFS/ME demonstrated in multiple studies. Response to rintatolimod demonstrated in patients. Alternatively, dimer/trimer (and above) ratios can be determined and quantified	Research methods of analysis are not suitable for clinical reference laboratories. Identification of 37kD/83kD forms should be amenable to mass spectrometry analysis as well as 2ʹ-5ʹA oligomers
Mitochondrial dysfunction	Assayed in PBMCs from a single laboratory in multiple reports. Observed in muscle by the same laboratory	Assay needs independent validation. Early ET oxygen debt experienced by CFS/ME patients supports data in PBMCs
Multiplex PCR analysis of mRNA and SNPs in literature identified genes associated with CFS/ME	Platforms supporting multiplex analysis for dozens of identified genes are available that allow simultaneous quantitative analysis of genes implicated in CFS/ME	Key genes can be identified with practical analysis using multiplex platforms available in clinical reference laboratories
Massive parallel sequencing	Identification of microorganisms in plasma and/or PBMCs by Next Generation Sequencing (NGS). Human sequences easily distinguished from non-human	New NGS platforms are currently reaching clinical reference laboratories with significant reduction in costs. Potential for identification of new pathogens responsive to rintatolimod. New SNPs identifiable

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