METHODS FOR TESTING OF THERAPEUTIC COMPOUNDS

Abstract: The present invention is related to a method for determining the efficacy of a chemical compound for treatment of recurrent herpes virus infection, said method comprising: (a) inoculating herpes virus at a primary skin site of a non-human mammal to cause zosteriform spread of the virus through nerve cells to cause lesions at a secondary (distal) site of infection; (b) before the appearance of skin lesions at the secondary site caused by the virus, giving adoptive transfer of immunity (ATI) to the said non-human mammal, so as to provide immunity mimicking the immunological reactivity seen in a recurrent herpes virus infection in a previously infected immune host; (c) administering a chemical compound to be tested to the said non-human mammal; and (d) determining the ability of the said chemical compound to reduce the occurrence and/or severity of herpes virus-induced skin lesions at the secondary site of infection.

Description: METHODS FOR TESTING OF THERAPEUTIC COMPOUNDS TECHNICAL HELD The present invention relates to methods for determining the efficacy of chemical compounds for treatment of recurrent herpes virus infection. BACKGROUND ART Herpes viruses, such as herpes simplex viruses (HSV), initially infect an individual through a primary infection. This infection is different from subsequent recurrent infections arising from reactivation of latent HSV from a number of aspects. A primary HSV infection affects an individual whose immune response has not previously encountered the virus. In the case of such a primary infection it therefore takes some time for the immune system to recognize the intruder and mount an immune response leading to an inflammation strong enough to eradicate the replicating virus in the body. A primary HSV infection, in comparison to a recurrent HSV infection, is therefore characterized by a longer lasting viral shedding and a less excessive inflammation caused by immune reactivity. Following a primary infection with herpes simplex virus or Varicella-zoster virus, the virus establishes latency in nerve cells. The virus can subsequently be reactivated repeatedly during the rest of the patient's life. Following a reactivation in the nerve cell, the virus is transported to the skin and subsequently a lesion develops. When reactivated herpes virus infections occur they will affect individuals who may have encountered the virus many times before and earlier have successfully eradicated the replicating virus. It is therefore not surprising that reactivated HSV infections in comparison to primary HSV infections are characterized by a shor ten-las ting viral shedding period and a rapid and substantial immune response causing inflammation. The inflammation is the main cause of the symptoms and signs which the patient will experience such as pain, itching, swelling, redness and lesions. The symptoms may last for approximately a week or longer after the end of the viral shedding period. In the case of a primary labial infection of herpes simplex virus, virus shedding occurs for 7 to 10 days. In the case of a primary genital infection with the same virus, virus " shedding occurs for around 3 weeks. (Whitley, R.J. and Gnann, J.W., In: The Human Herpesviruses (Ed: B. Roizman, R.J. Whitley and C. Lopez) Raven Press, New York, 1993) Following termination of viral shedding the lesions will heal in a few days. In the case of both labial and genital recurrent HSV infections, on the other hand, virus shedding occurs on average only over 3 to 4 days (Spruance, S.L. (1992) Seminars in Dermatology 11, 200-206; Vanderhooft, S and Kirby, P. (1992) Seminars in Dermatology 11, 190-199). As discussed above, the lesions do not heal rapidly following termination of virus shedding due to the substantial inflammation. A typical labial or genital lesion resulting from recurrent HSV infection will heal in around 10 days. Because of the longer viral shedding period in primary infections due to the time requirement to develop a potent immune reactivity, clinical studies with antiviral compounds show substantially larger effect against a primary infection in comparison to recurrent infections. In the case of recurrent HSV infections at best, only around one day improvement in healing time and viral shedding duration is shown in clinical trials, even when the antiviral drugs are administered systemicly (Spruance, S.L. (1992) Seminars in Dermatology 11, 200-206; Whitley, R.J. and Gnann, J.W. (1992) New England Journal of Medicine 327, 782-789). Reliable animal models are essential for the development of antiviral compounds. A number of primary infection models have been published. From the difference in pathogenesis of primary and recurrent herpes virus infections described above, it is dear that results from primary infection models cannot readily be used as an indicator to predict clinical efficacy against recurrent herpes virus infections. Recurrent HSV models to evaluate antiviral compounds have been developed in guinea-pigs (Stanberry, L.R. (1989) J. Med. Virol. 28: 125-128). In the model of Stanberry and coworkers, HSV-2 is inoculated intravaginally in to the guinea-pig. After 100 days, the animals are subjected to UV radiation to induce a reactivation of the latent virus. Around 70% of the animals will then develop a lesion with a median duration of 1-2 days. This model is tύ-ne-consuming and expensive, but the main concern with regard to clinical relevance is the short lasting duration of the lesion. In comparison, human recurrent HSV lesions last for 7-12 days. The Stanberry model can thus be used only for assessment for prophylactic treatment. Treatment initiated after the onset of symptoms cannot be assessed in this model due to the short lasting lesion duration. Another approach pioneered by Blyth et al. (J. gen. Virol. (1984), 65, 1477- 1486) is based on the zosteriform spread of herpes viruses. After primary infection of the skin by a herpes virus (with subsequent lesion formation at the site of primary infection), the virus enters and is transported along the peripheral nerves. Through this transport of the virus in the nerves, secondary lesions can occur at a secondary site of infection distal to the original primary site of infection. This manner of spread of the virus infection is termed zosterifom spread. Two such models have been developed: (i) the flank model, wherein virus is inoculated in the flank and a zosteriform spread is noted along the affected dermatome of the flank four to five days later; and (ϋ) the neck-ear model, wherein virus is inoculated in the neck (the primary site) and the virus spreads to the corresponding ear (the seconday distal site) four to five days later. Even though the lesion duration in these models is similar to the clinical situation, the infection is still a primary infection, since the animal has not previously encountered the virus. It is well known that if an animal has been previously inoculated with the virus, a second attempt at infection will not result in lesions, either at the site of infection, or by zosteriform spread. Consequently, such models can be regarded as primary infection models. The mode of infection in human recurrent HSV infection and murine zosteriform HSV infection is similar i.e. entry of virus into the skin via sensory nerves. However, in man, recurrent herpetic disease appears in an immune individual, whereas in the zosteriform infection models, immunity does not exist at the time of infection but is developed during the course of infection. The preclinical assessment of drugs intended for use on recurrent herpes virus infection in immune individuals is therefore problematic. There is a need for an animal model which mimics the inflammatory response and the eradication of recurrent herpes virus infection in an immune individual and thereby provides a means for predicting the clinical effect of chemical compounds against recurrent, not only primary, herpes virus infections. It should furthermore be possible in such a model to administer the test compounds following initiation of symptoms and not only prophylactically. PURPOSE OF THE INVENTION It has surprisingly been found that adoptive transfer of immunity (ATI) can be provided after inoculation of an animal with HSV without compromising the incidence of subsequent zosteriform spread and secondary lesion appearance. The above-mentioned problems with the previously known infection models are therefore solved, according to the present invention, by providing a method for determining the efficacy of a chemical compound against progress of recurrent herpes virus infection, characterized by the effect of the test compound being determined by assessing the progress of infection in a non-human mammal which has been given adoptive transfer of immunity (ATI) in order to mimic the immunological reactivity of an immune host. In an animal model according to the present invention, zosteriform secondary lesions appear 4-5 days following primary inoculation of herpes virus. The virus shedding is short-lasting (around 3 to 4 days), followed by a longer-lasting inflammation causing the clinical symptoms. Swelling is one of the main characteristics of inflammation and is easily measurable in the case of a neck-ear model of the present invention by recording the thickness of the ears. Furthermore, such an animal model, has an effective immune response towards the virus at the time of arrival of the virus at the secondary site of infection (The ear). The model thus mimics the clinical situation. When UV-inactivated virus was injected into the ear of, for example a mouse, modification of immunity by ATI, before the virus reaches the skin of the test animal, potentiates the immune response. Zosteriform spread of HSV in animals given ATI is therefore believed to closely mimic the pathogenesis of human recurrent HSV infection. An animal model of the invention which has been inoculated with HSV in the neck and human recurrent HSV infection show the following measurable similarities: (i) a similar length of viral shedding (around 3 to 4 days); (ii) the maximum virus litre is obtained 24 hours following the first appearance of virus in the ear; (iii) a similar reduction of viral shedding (1 day) when topical foscamet is used for treatment; (iv) as in the clinical situation, the immune response causing inflammation appears to be the key mediator of most symptoms. If +ATI animals according to the invention inoculated with HSV in the neck are compared to -AΗ animals, similarly inoculated with HSV the animals with immune cells present at the time of arrival of virus to the ears exhibit stronger inflammation (as measured by ear thickness) and a worse clinical picture (as measured by zoster score) even though the virus is cleared much more rapidly by the +ATI animals (Fig. 2). As indicated above, this is also paralleled by the clinical picture of recurrent herpes virus infections in humans. This clearly shows that the ATI procedure utilized in a model of the invention is critical for good prediction of clinical efficacy in treating such an infection. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 Effects of the developing immunity and ATI. Virus was inoculated on the back of the animal on day zero. AΗ was given on day two. The immune response against HSV was tested by injection of UV-inactivated HSV-1 into the ear pinna on day 2. Results are the median values of differences in ear thickness between right and left pinna in 15 Balb/C mice. Fig. 2 Effects of ATI and topical treatment with foscamet on HSV-1 titers in the ear pinna of neck-infected Balb/C mice, (n=10). ATI was given on day two. Fig. 3 Effects of topical treatment with foscamet on the development of ear lesions in neck-infected Balb/C mice (n=12). ATI was given two days post- neck infection. Treatment was given every 8 h. day 4 to day 7 post- infection. Fig. 4 Effects of ATI on the development of ear lesions of neck-infected Balb/C mice, (n=12). Placebo was given every 8 h. day 4 to day 8 post-infection. Left panel, no ATI given; Right panel, ATI given 2 days post-infection. Fig. 5 Effects of ATI on ear swelling of neck-infected Balb/C mice, (n=12). Placebo was given every 8 h. day 4 to day 8 post-infection. Left panel, no ATI given; Right panel, ATI given 2 days post-infection. DISCLOSURE OF THE INVENTION The present invention provides a method for determining the efficacy of a chemical compound for treatment of recurrent herpes virus infection, comprising (a) inoculating herpes virus at a primary skin site of a non-human mammal to cause zosteriform spread of the virus through nerve cells to cause lesions at a secondary (distal) site of infection; (b) before the appearance of skin lesions at the secondary site caused by the virus, giving adoptive transfer of immunity (ATI) to the said non-human mammal, so as to provide immunity mimicking the immunological reactivity seen in a recurrent herpes virus infection in a previously infected immune host; (c) administering a chemical compound to be tested to the said non- human mammal; and (d) determining the ability of the said chemical compound to reduce the occurrence and/or severity of herpes virus-induced skin lesions at the secondary site of infection. The term "herpes virus" should be understood to refer to the group of viruses comprising herpes simplex virus type 1 (HSV-1); herpes simplex virus type 2 (HSV-2); Human herpes virus type 6 (HHV-6); Varicella zoster-virus (VZV); cytomegalovirus (CMV); and Epstein-Barr virus (EBV). Preferably, the herpes virus employed for inoculation in a method of the invention is a herpes virus of the α-group, such as herpes simplex virus type 1 or type 2, or Varicella-zoster virus. It will be understood, however, that also other types of herpes viruses, which may be yet unknown, can be alternatively used. The infectious agent can be administered to the non-human mammal by inoculation of the ventral surface of the neck. The progression of infection is then assessed by inspection of the occurrence of lesions in the ear pinna (neck-ear model). Alternatively, the infectious agent can be administered to the non-human mammal by inoculation of the dorsal flank. In this case, the progression of infection is assessed by inspection of the severity of lesions on the skin (flank model). The non-human mammal to be used in a method according to the invention is preferably a laboratory animal such as a mouse, a rat, a rabbit or a guinea-pig. In a method according to the invention, provision of adoptive transfer of immunity in the animal model may preferably comprise injecting into the chosen non-human mammal for said model lymph node cells obtained by a method comprising (a) administering the herpes virus to a second non- human mammal of the same inbred strain as the animal model; and (b) after the said second non-human mammal has established effective immunity against the virus, collecting lymph node cells from that mammal. The skilled person will be able to determine when such effective immunity has been established in the second non-human mammal. In normal cases, the period for the animal to develop effective immunity will be from 1 week to 1 month. Inoculation of lymph node cells to provide AΗ should be performed after HSV infection has been established in the nerves, but before the virus reaches the secondary site of infection. Preferably, ATI is performed 1 to 4 days, more preferably approximately 2 days, after infection. The number of lymph node cells injected into the non-human mammal chosen for the animal model can vary within a wide range, provided that the number is sufficient to produce immunity in the target animal. The said number of cells can e.g. be from 10 6 to 10 . The compound to be tested can be administered to the non-human mammal in any suitable way, e.g. topically, orally or by injection. The invention also comprises a non-human mammal, e.g. a mouse, a rat, a rabbit or a guinea-pig, which animal is deliberately infected with a herpes virus, and which animal has been given adoptive transfer of immune cells (ATI) in order to mimic the immunological reactivity of an immune host. The said herpes virus is preferably a herpes simplex virus type 1 or type 2, or a Varicella-zoster virus. EXAMPLES 1. Materials and methods 1.1. Inoculation of mice Two models of zosteriform spread HSV-1 infections were used to study the efficacy of the various treatments. In the neck-ear-model virus is inoculated into the ventral surface of the neck (primary site), which causes zosteriform spread of HSV-1 to the corresponding ear pinna and swelling of the ear (secondary site). In the flank model virus is inoculated into the dorsal flank just lateral to the spine (primary site), giving rise to a zosteriform rash within the infected dermatome at the lower flank (secondary site). Human HSV-1 (2 x 10 5 p.f.u., strain C42) was inoculated into the neck or back of groups of 10-15 female Balb/C mice (16-18 g) as described in Kristofferson et al. (1988) J. gen. Virol. 69, 1157-1166. The development of zosteriform spread infection was recorded by daily inspection of the occurrence of lesions on the pinna or, for flank infections, by scoring the severity of lesions on the skin according to the following scale: -4 no symptoms; -3 one primary vesicle and swelling; -2 some primary vesicles; -1 local erosion; 0 ulceration of the local primary lesion; 1 primary lesion plus isolated zosteriform secondary lesions. Scores 2-4 describe the ulceration of confluent zosteriform lesions from mild to severe (Nagafuchi, S. et al. (1979) J. gen. Virol. 44, 715-723). Thus, scores 1 to 4 describe the development of the zosteriform lesion (zoster score). In neck-infected mice, ear swelling and titers of infectious virus in ears were recorded as described by Kristofferson et al. (1988) J. gen. Virol. 69, 1157-1166. 10 μl UV-inactivated HSV-1, corresponding to 10 5 p.f.u., was injected intradermally into the centre of the back of the pinna of the right ear of back-infected mice 2 days p.i.. 1.2. Adoptive transfer of HSV-immunity (AΗ) HSV-1 (10 p.f.u., strain C42) was injected into the pinna of both ears of anaesthetized female Balb/C mice (16-18 g). Seven days p.i. the animals were sacrificed by cervical dislocation, the draining lymph nodes were removed, and a suspension of lymph node cells in phosphate buffered saline was prepared by means of a micromesh. n When giving ATI, 2 x 10 lymph node cells were injected intravenously into the tail of neck-or back-infected mice 2 days pi.. 1.3. Drug treatment Foscamet σeam (1.5%, Astra Arcus AB, Sδdertalje, Sweden) was prepared as described by Alenius et al. (1982) J. Infectious Diseases 145, 569-573. The cream base without drug was used as placebo. 2. Results 2.1. Adoptive transfer of immunity (AΗ) Three or four days after inoculation of the skin of the ventral side of the neck with HSV-1, virus was detected in the ipsilateral ear. Virus titers increased for one day, but from 5-6 days p.i. and onwards virus was cleared. However, ear swelling in response to UV-inactivated HSV-1 (Fig. 1), injected into the pinna of back-infected mice at 2 days p.i., was not developed until day 5 p.i. (i.e. one to two days after entry of virus into the pinna of neck-infected mice). In back-infected mice given ATI 2 days p.i., ear swelling in response to UV-inactivated HSV-1 injected into the pinna 2 days p.i. occurs within 24 hours (Fig. 1). Therefore, in neck-infected mice given ATI at 2 days p.i., virus enters the pinna of animals capable of developing an immune response to HSV-1 antigens. ATI decreased virus titers in the ear of neck-infected animals (Fig. 2). However, ear swelling was not prevented (Fig. 5). The zoster score of the ears of neck-infected animals was increased by AΗ (Fig. 4). Neck-infected animals given ATI showed a stronger, more rapid and longer lasting swelling of the ears compared to animals not given AΗ (Fig. 5). Swelling is believed to correspond to inflammation and it is apparent that ATI worsens inflammation and zoster score even though virus is much more rapidly cleared. 2.2. Topical fosca et Ears of neck-infected mice, not given ATI, were treated with topical 1.57o foscamet or placebo cream starting 4-days p.i. and continuing for 3 days. Virus titers in the ear and clinical signs were recorded. It is shown in Fig. 2 that foscarnet-treatment of animals not given AΗ decreases virus titers substantially, in particular on day 7 and 8 after infection. Foscamet treatment of animals given AΗ showed only a minor effect where the viral shedding period was only reduced by one day. This is similar to the clinical experience of foscarnet-treatment of patients with recurrent HSV infection. This effect was paralleled in the assessment of the fraction (%) of ATI-treated animals showing clinical lesions (Fig. 3) after treatment with placebo or foscamet. The results indicate that some effect can be obtained with foscamet treatment in animals given ATI.

Claims: CLAIMS 1. A method for determining the efficacy of a chemical compound for treatment of recurrent herpes virus infection, comprising (a) inoculating herpes virus at a primary skin site of a non-human mammal to cause zosteriform spread of the virus through nerve cells to cause lesions at a secondary (distal) site of infection; (b) before the appearance of skin lesions at the secondary site caused by the virus, giving adoptive transfer of immunity (ATI) to the said non-human mammal, so as to provide immunity mimicking the immunological reactivity seen in a recurrent herpes virus infection in a previously infected immune host; (c) administering a chemical compound to be tested to the said non- human mammal; and (d) determining the ability of the said chemical compound to reduce the occurrence and/or severity of herpes virus-induced skin lesions at the secondary site of infection. 2. A method according to claim 1 wherein the herpes virus employed for inoculation is herpes simplex virus type 1 or type 2. 3. A method according to claim 1 wherein the herpes virus employed for inoculation is Varicella-zoster virus. 4. A method according to any one of claims 1 to 3 wherein the herpes virus is administered to the non-human mammal by inoculation in the ventral surface of the neck and wherein the progression of infection is assessed by inspection of the occurrence of lesions in the pinna. A method according to any one of claims 1 to 3 wherein the herpes virus is administered to the non-human mammal by inoculation in the dorsal flank and wherein the progression of infection is assessed by inspection of the severity of lesions on the skin. 6. A method according to any one of claims 1 to 5 wherein the said non-human mammal is a mouse, a rat, a rabbit or a guinea-pig. 7. A method according to any one of claims 1 to 6 wherein said adoptive transfer of immunity is provided in said non-human mammal 1 to 4 days after inoculation of the herpes virus. 8. A method as claimed in claim 7 wherein said adoptive transfer of immunity is provided in said non-human mammal about 2 days after inoculation of the herpes virus. 9. A method according to any one of claims 1 to 8 wherein the said adoptive transfer of immunity is provided by injecting lymph node cells collected from a second mammal which has developed immunity to said herpes virus, said second mammal and said mammal given adoptive transfer of immunity being of the same inbred strain. 10. A method according to claim 9 wherein the number of lymph node cells being injected into the non-human mammal is from 10 6 to 10 .

Inventors: KRISTOFFERSON ANN, KRISTOFFERSON, ANN
Applicants: ASTRA AB, KRISTOFFERSON ANN, ASTRA AKTIEBOLAG, KRISTOFFERSON, ANN
Application year: 1996
Main classification: C12Q1
IPC codes: A61K49, C12Q1, G01N33