Treatment of diseases

[0040] Example of production of goat serum

[0041] Goats were inoculated by intramuscular injection of a lysed HIV virus mixture in Freund's adjuvant. The virus was first heat killed at 60°C for 30 minutes. After an appropriate time interval, such as 2 weeks, blood samples are drawn for initial evaluation. In the optimized protocol, goats were injected weekly for 4 weeks, and then blood was drawn from the animals at 6 weeks to obtain the agent.

[0042] Under aseptic technique, approximately 400 cc of blood was extracted from the goat. The area for needle extraction was shaved and prepared with povidone-iodine. An 18-gage needle was used to extract approximately 400 cc of blood from the animal. It should be noted that animals tolerated the withdrawal of approximately 400 cc of blood without suffering any adverse effects. The animals do not have to be sacrificed. The animal can then be bled again approximately 10-14 days after it has replenished its blood volume.

[0043] The presence of potentially potent antibodies was confirmed, noting the desired antibody activity. Once the presence of such agents was confirmed, blood was then drawn from the goats between 4-6 weeks.

[0044] To produce the reagents, the base blood product is then centrifuged to produce serum. Then 300ml of serum was filtered to remove large clots and particulate matter. The serum was then treated with supersaturated ammonium sulfate (45% solution to room temperature) to precipitate antibodies and other substances. The solution thus obtained was centrifuged at 5000 rpm for 5 minutes, after which the supernatant fluid was removed. The precipitated immunoglobulin was resuspended in phosphate-buffered saline (PBS buffer, see Sambrook et al., 'Molecular Cloning: A Laboratory Manual', 1989), and the precipitate was sufficiently redissolved.

[0045] The solution was then dialyzed through a membrane with a molecular weight cut off of 10,000 Daltons. Dialysis was performed in PBS buffer, which was changed every 4 hours over the course of 24 hours. Dialysis was performed at 4°C.

[0046] After 24 hours of dialysis, completely decant the contents of the dialysis bag into a sterile beaker. The solution was adjusted so that the mass per unit volume = 10 mg/ml. Dilutions were performed with PBS. The solution thus obtained was then filtered through a 0.2 micron filter into sterile containers. After filtration, the solution was aliquoted into single doses of 1 ml and stored at -22°C until application. The composition is referred to herein as AIMSPRO serum.

[0047] neurological disease

[0048] Acute optic neuritis is a common manifestation of multiple sclerosis. It presents as an onset of monocular blurring in central vision with a pronounced effect on color discrimination. Successive attacks can lead to irreversible and often slowly progressive, vision loss, although spontaneous resolution is usually followed 1. No drug is available to improve visual function in patients with these chronic effects. Here we provide evidence of a promising therapeutic avenue together with an electrophysiological indication of the remarkable rapidity of onset.

[0049] Six patients with multiple sclerosis (2 males, 4 females, aged 32-42 years, disease duration 8-16 years) with stable visual dysfunction caused by chronic optic neuritis were treated with a product called Aimspro For treatment, the Aimspro was obtained from purified goat serum as described above and in WO03/004049 and WO03/064472. The drug is administered in 1 ml by subcutaneous injection, usually self-administered after the first or second dose. The frequency of administration, adjusted by response, varies from once to three times per week. No patient had previously received the product, but one patient (case 2) had been taking interferon beta-1a (Rebif) for almost a year: this treatment was discontinued the day before treatment with Aimspro. Recordings were made immediately before the first injection, and approximately 1 hour, 1 week, and 4-7 weeks after the injection. Before treatment, all subjects described that their vision had slowly and gradually deteriorated over a period of 3-14 years, and none could recall an intervening period that could represent acute optic neuritis.

[0050] Corrected distance acuity (Snellen chart) and color vision (square root of full error scores from Farnsworth-Munsell 100-Hue test) obtained under standardized lighting conditions are listed 2 )data sheet). Monocular visual evoked potential (VEP) studies were performed in each case. Perimetry was not performed. Sublingual temperature was monitored over time in subjects and did not show significant variability. Data for the left and right eyes were considered independent for analysis, and for statistical purposes the color vision scores were treated as non-parametric.

[0051] Comparing pretreatment and follow-up distance acuity did not show significant changes, and only in two eyes (case 2 left eye and case 5 right eye) were there one or more lines on the Snellen chart improve. However, a repeated measures analysis of variance (ANOVA) test on color vision scores yielded F = (2.16, 23.73) = 8.52, p = 0.001. Within approximately 1 hour of injection, there was a significant improvement in color vision (p=0.008, Z=-2.667, Wilcoxon signed ranks test). Comparing pre-treatment and "one week" values ​​showed no significant difference (p=0.055, 2=-1.923), but comparison of pre-treatment and follow-up data (at 4-7 weeks) showed a significant benefit (p=0.003, Z=-2.981). Except for local pain and swelling at the injection site during the first 2-3 weeks, no significant side effects were encountered in 3 patients.

[0052] For cases 5 and 6, VEP response latencies were at the upper limit of normal. Pretreatment VEP studies from all remaining eyes except one showed delays in P100 responses consistent with demyelination within the visual pathway. In only one instance (Case 4 right eye) was there no response available prior to treatment, and this was the only eye in the entire series that showed a significant change in mean cortical response at any time during the observation period. This 42-year-old woman, who had a secondary spinal multiple sclerosis attack in 1992, complained of progressively worsening vision since 1998. Between 1993 and 1997 there had been 4 cycles of blurred visual resolution of 3-7 day duration, but more recent onset visual features were absent in the medical records. Examination revealed bilateral optic atrophy and marked impairment of distance and color vision. Pre-treatment full field pattern reversal VEP studies at 15:02 hrs did not produce reproducible traces from the right eye (see Figure 1). A test dose of Aimspro (0.1 ml) was administered subcutaneously at 15:13 hrs, followed by an additional 0.9 ml at 15:25 hrs. A significant but reproducible P100 response is now available at 15:43 hrs, at 171 ms (see Figure 2). Scalp leads remained attached throughout the study, and test conditions, including body temperature, were monitored. Although this neurophysiological finding is consistent with a reversal of the transduction block in severely demyelinated fibers 3 , but it was not accompanied by a clinically significant improvement in the sensitivity data. The fact that no improvement in P100 latency was detectable from any eye during the study period shows the opposite conclusion that there was significant remyelination during this time period, but additionally, observations at approximately 6 months will be required to fully assess this fact.

[0053] In summary, non-blinded, uncontrolled observations in 6 patients with slowly progressive visual dysfunction due to optic neuritis, after 4-7 weeks of treatment with the novel drug Aimspro Significant improvement in color vision was shown during the course of treatment. Neurophysiological data from one infected eye of a patient with a 5-year history of significant visual deficits are consistent with the interpretation that drug administration caused a reversal of axonal conduction block. And, although this phenomenon was shown to occur within 30 minutes of treatment, the authors' clinical observations (unpublished observations) in a 38-year-old female patient with relapsing-remitting multiple sclerosis with "spinal" relapses, Indicating that "unblocking" can be seen in as little as 10 minutes. Further observations (unpublished observations) in a patient with 18 years of stable motoron loss associated with severe acute infectious polyneuritis (Guillain-Barresyndrome) suggest that the effects may also be related to the peripheral nervous system.

[0054] Visual deficits (as measured by clinical and neurophysiological examination) in acute optic neuritis are thought to reflect axonal conduction block associated with local inflammatory demyelinating activity 4，5，6 , but inflammation seems unlikely to be a persistent factor in cases of chronic infection such as the 6 patients described above. Therefore, the direct action of the drug ingredients on neurotransmitters is doubtful. With a view to elucidating the mechanism of action, basic neurophysiological techniques are now applied.

[0055] Aimspro is a serum product originally intended to provide high-titer neutralizing antibodies for use in HIV patients. The sera were characterized by high titers of anti-HLA2 class antibodies capable of suppressing various mixed lymphocyte responses (unpublished observation). Since increased HLA2 class expression on brain cells and lymphocytes is thought to be a major factor in the inflammatory process in multiple sclerosis, it can be hypothesized that polyclonal serum might be beneficial in multiple sclerosis and similar conditions (see ref. Literature 1). In fact, monoclonal antibodies against the HLA2 class are under development by some companies. However, the rapidity of the clinical response observed here suggests that other mechanisms may be at play in vivo. Delay in sodium channel inactivation, and blockade of potassium channels both shown to improve transduction in experimentally demyelinated axons 7. Alternatively, by endogenous substances such as nitric oxide 8 Removing the blockade of axonal sodium channels could explain the rapidity of drug action. Therefore, it is possible that besides some role said serum has in influencing immune events, it may also directly affect the safety of axonal conduction.

[0056] autoimmune disease

[0057] The Aimspro products can also be used in the treatment of autoimmune diseases, as described below. 1 ml of aliquot serum prepared as described was adjusted to provide a dose of 0.1 mg/kg, and subcutaneously injected into patients with autoimmune diseases selected from the group consisting of lupus, psoriasis, eczema, thyroiditis, and Polymyositis.

[0058] The product is administered to the patient as described below. A male patient with psoriasis begins with an initial presentation that begins on the hands but extends over most of the lower legs and arms. The treating physician prescribed chlorphenamine, then mometasone. By the end of the month, the condition is more widely distributed. Prescribed Polytar softener and consulted with a consultant dermatologist who confirmed psoriasis and prescribed mometasone, Polytar and Exorex. The treatment had little effect and the psoriasis was worst on the arms and legs. Start AIMSPRO product on day 1, 1ml per week. On day 5, the psoriasis started to improve. On day 23, exfoliation improved even more. Increase dose to 2amps per week. After 2 months the patient improved even more and by 3 months and 18 days the psoriasis is now considered cured and the patient wishes to stop the treatment. This gives 1 amp per week for 4/52 and 2 amps per week for 12/52; a total of 16 weeks of 28 amps. There were no reported side effects.

[0059] references.

[0060] 1. Compston A, Coles A. Multiple Sclerosis, Lancet 2002;359:1221-31

[0061] 2. Farnsworth D. The Farnsworth-Munsell 100-Hue and Dichotomous Tests for Color Vision. J Opt Soc Am, 33, 568 (1943).

[0062] 3. McDonald WI, Sears TA. The effect of experimental demyelination on conduction in the central nervous system. Brain 1970; 93, 583-598.

[0063] 4. Hawkins CP, et al. Duration and selectivity of blood-brain barrier breakdown in chronic relapsing experimental allergic encephalomyelitis studied by gadolinium-DTPA and protein markers. Brain 1990; 113, 365-378.

[0064] 5. Katz D, Taubenberger J, Raine C, McFarlin D, McFarland H. Gadolinium-enhancing lesions on magnetic resonance imaging. Ann Neural 1990; 28, 243.

[0065] 6. Youl BD, et al The pathophysiology of acute optic neuritis: an association of gadolinium leakage with clinical and electrophysiological deficits. Brain1991; 114; 2437-2450.

[0066] 7. Smith KJ, McDonald WI. The pathophysiology of MS: the mechanisms underlying the production of symptoms and the natural history of disease. Philos Trans R Soc Lond B Biol Sci 1999; 354: 1649-1673.

[0067] 8. Redford EJ, Kapoor R and Smith KJ. Nitric oxide donors reversibly blockaxonal conduction: demyelinated axons are especially susceptible. BrainPart 12(Dec 1997) 2149-57.

[0068] Table legend:

[0069] MS Type SP (Secondary) RR (Relapsing Remitting)

[0070] Dx yrs years from multiple sclerosis likely onset

[0071] VsDtn yrs years of progressive vision loss

[0072] EYE Right eye and left eye are treated independently

[0073] P100ms P100 VEP positive latency in milliseconds

[0074] VApre Derived from the Snellen Vision Chart for Visual Acuity Pretreatment

[0075] VA 1 hour As above, approximately 1 hour after treatment

[0076] VA 7 days As above, within 7 days

[0077] VA FU as above, at follow-up (4-7 weeks)

[0078] Square root of CV pre Farnsworth-Munsell 100-Hue test

[0079] CV 1 hour As above, approximately 1 hour after treatment

[0080] CV 7 days As above, at 7 days

[0081] CV FU as above, at follow-up

[0082] NR no response

[0083] Demographics, psychophysics, neurophysiology data

[0084] MS